Details of a Researcher - Saya, Hideyuki

Papers - Saya, Hideyuki

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2-Nitroimidazoles induce mitochondrial stress and ferroptosis in glioma stem cells residing in a hypoxic niche

Koike N., Kota R., Naito Y., Hayakawa N., Matsuura T., Hishiki T., Onishi N., Fukada J., Suematsu M., Shigematsu N., Saya H., Sampetrean O.

Communications Biology (Communications Biology) 3 ( 1 ) 2020.12

© 2020, The Author(s). Under hypoxic conditions, nitroimidazoles can replace oxygen as electron acceptors, thereby enhancing the effects of radiation on malignant cells. These compounds also accumulate in hypoxic cells, where they can act as cytotoxins or imaging agents. However, whether these effects apply to cancer stem cells has not been sufficiently explored. Here we show that the 2-nitroimidazole doranidazole potentiates radiation-induced DNA damage in hypoxic glioma stem cells (GSCs) and confers a significant survival benefit in mice harboring GSC-derived tumors in radiotherapy settings. Furthermore, doranidazole and misonidazole, but not metronidazole, manifested radiation-independent cytotoxicity for hypoxic GSCs that was mediated by ferroptosis induced partially through blockade of mitochondrial complexes I and II and resultant metabolic alterations in oxidative stress responses. Doranidazole also limited the growth of GSC-derived subcutaneous tumors and that of tumors in orthotopic brain slices. Our results thus reveal the theranostic potential of 2-nitroimidazoles as ferroptosis inducers that enable targeting GSCs in their hypoxic niche.

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Phosphorylation of the Anaphase Promoting Complex activator FZR1/CDH1 is required for Meiosis II entry in mouse male germ cell

Tanno N., Kuninaka S., Fujimura S., Takemoto K., Okamura K., Takeda N., Araki K., Araki M., Saya H., Ishiguro K.i.

Scientific Reports (Scientific Reports) 10 ( 1 ) 2020.12

© 2020, The Author(s). FZR1/CDH1 is an activator of Anaphase promoting complex/Cyclosome (APC/C), best known for its role as E3 ubiquitin ligase that drives the cell cycle. APC/C activity is regulated by CDK-mediated phosphorylation of FZR1 during mitotic cell cycle. Although the critical role of FZR1 phosphorylation has been shown mainly in yeast and in vitro cell culture studies, its biological significance in mammalian tissues in vivo remained elusive. Here, we examined the in vivo role of FZR1 phosphorylation using a mouse model, in which non-phosphorylatable substitutions were introduced in the putative CDK-phosphorylation sites of FZR1. Although ablation of FZR1 phosphorylation did not show substantial consequences in mouse somatic tissues, it led to severe testicular defects resulting in male infertility. In the absence of FZR1 phosphorylation, male juvenile germ cells entered meiosis normally but failed to enter meiosis II or form differentiated spermatids. In aged testis, male mutant germ cells were overall abolished, showing Sertoli cell-only phenotype. In contrast, female mutants showed apparently normal progression of meiosis. The present study demonstrated that phosphorylation of FZR1 is required for temporal regulation of APC/C activity at meiosis II entry, and for maintenance of spermatogonia, which raised an insight into the sexual dimorphism of FZR1-regulation in germ cells.

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Downregulation of the CCL2/CCR2 and CXCL10/CXCR3 axes contributes to antitumor effects in a mouse model of malignant glioma

Shono K., Yamaguchi I., Mizobuchi Y., Kagusa H., Sumi A., Fujihara T., Nakajima K., Kitazato K.T., Matsuzaki K., Saya H., Takagi Y.

Scientific Reports (Scientific Reports) 10 ( 1 ) 2020.12

© 2020, The Author(s). Glioblastoma multiforme involves glioma stem cells (GSCs) that are resistant to various therapeutic approaches. Here, we studied the importance of paracrine signaling in the glioma microenvironment by focusing on the celecoxib-mediated role of chemokines C–C motif ligand 2 (CCL2), C-X-C ligand 10 (CXCL10), and their receptors, CCR2 and CXCR3, in GSCs and a GSC-bearing malignant glioma model. C57BL/6 mice were injected with orthotopic GSCs intracranially and divided into groups administered either 10 or 30 mg/kg celecoxib, or saline to examine the antitumor effects associated with chemokine expression. In GSCs, we analyzed cell viability and expression of chemokines and their receptors in the presence/absence of celecoxib. In the malignant glioma model, celecoxib exhibited antitumor effects in a dose dependent manner and decreased protein and mRNA levels of Ccl2 and CxcL10 and Cxcr3 but not of Ccr2. CCL2 and CXCL10 co-localized with Nestin+ stem cells, CD16+ or CD163+ macrophages and Iba-1+ microglia. In GSCs, celecoxib inhibited Ccl2 and Cxcr3 expression in a nuclear factor-kappa B-dependent manner but not Ccr2 and CxcL10. Moreover, Ccl2 silencing resulted in decreased GSC viability. These results suggest that celecoxib-mediated regulation of the CCL2/CCR2 and CXCL10/ CXCR3 axes may partially contribute to glioma-specific antitumor effects.

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Molecular and cellular mechanisms underlying brain metastasis of breast cancer

Hosonaga M., Saya H., Arima Y.

Cancer and Metastasis Reviews (Cancer and Metastasis Reviews) 39 ( 3 ) 711 - 720 2020.09

ISSN 01677659

© 2020, The Author(s). Metastasis of cancer cells to the brain occurs frequently in patients with certain subtypes of breast cancer. In particular, patients with HER2-positive or triple-negative breast cancer are at high risk for the development of brain metastases. Despite recent advances in the treatment of primary breast tumors, the prognosis of breast cancer patients with brain metastases remains poor. A better understanding of the molecular and cellular mechanisms underlying brain metastasis might be expected to lead to improvements in the overall survival rate for these patients. Recent studies have revealed complex interactions between metastatic cancer cells and their microenvironment in the brain. Such interactions result in the activation of various signaling pathways related to metastasis in both cancer cells and cells of the microenvironment including astrocytes and microglia. In this review, we focus on such interactions and on their role both in the metastatic process and as potential targets for therapeutic intervention.

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Targeting of cancer stem cells by differentiation therapy

Arima Y., Nobusue H., Saya H.

Cancer Science (Cancer Science) 111 ( 8 ) 2689 - 2695 2020.08

ISSN 13479032

© 2020 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association. Chemoresistance is a hallmark of cancer stem cells (CSCs). To develop novel therapeutic strategies that target CSCs, we established osteosarcoma-initiating (OSi) cells by introducing the c-Myc gene into bone marrow stromal cells derived from Ink4a/Arf KO mice. These OSi cells include bipotent committed cells (similar to osteochondral progenitor cells) with a high tumorigenic activity as well as tripotent cells (similar to mesenchymal stem cells) of low tumorigenicity. We recently showed that the tripotent OSi cells are highly resistant to chemotherapeutic agents, and that depolymerization of the actin cytoskeleton in these cells induces their terminal adipocyte differentiation and suppresses their tumorigenicity. We here provide an overview of modulation of actin cytoskeleton dynamics associated with terminal adipocyte differentiation in osteosarcoma as well as discuss the prospects for new therapeutic strategies that target chemoresistant CSCs by inducing their differentiation.

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The insulin-PI3K-Rac1 axis contributes to terminal adipocyte differentiation through regulation of actin cytoskeleton dynamics

Kunitomi H., Oki Y., Onishi N., Kano K., Banno K., Aoki D., Saya H., Nobusue H.

Genes to Cells (Genes to Cells) 25 ( 3 ) 165 - 174 2020.03

ISSN 13569597

© 2020 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd Adipocyte differentiation is accompanied by a pronounced change in the actin cytoskeleton characterized by the reorganization of filamentous (F)-actin stress fibers into cortical F-actin structures. We previously showed that depolymerization of F-actin stress fibers induced by inactivation of RhoA–ROCK (Rho-associated kinase) signaling acts as a trigger for adipocyte differentiation. The relevance and underlying mechanism of the formation of cortical F-actin structures from depolymerized actin during adipocyte differentiation have remained unclear, however. We have now examined the mechanistic relation between actin dynamics and adipogenic induction. Transient exposure to the actin-depolymerizing agent latrunculin A (LatA) supported the formation of adipocyte-associated cortical actin structures and the completion of terminal adipocyte differentiation in the presence of insulin, whereas long-term exposure to LatA prevented such actin reorganization as well as terminal adipogenesis. Moreover, these effects of insulin were prevented by inhibition of phosphatidylinositol 3-kinase (PI3K)–Rac1 signaling and the actin-related protein 2/3 (Arp2/3) complex which is a critical component of the cortical actin networks. Our findings thus suggest that the insulin-PI3K-Rac1 axis leads to the formation of adipocyte-associated cortical actin structures which is essential for the completion of adipocyte differentiation.

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Gene Therapy Using Neural Stem/Progenitor Cells Derived from Human Induced Pluripotent Stem Cells: Visualization of Migration and Bystander Killing Effect

Tamura R., Miyoshi H., Morimoto Y., Oishi Y., Sampetrean O., Iwasawa C., Mine Y., Saya H., Yoshida K., Okano H., Toda M.

Human Gene Therapy (Human Gene Therapy) 31 ( 5-6 ) 352 - 366 2020.03

ISSN 10430342

© Copyright 2020, by Mary Ann Liebert, Inc., publishers 2020. Glioblastoma is the most aggressive brain tumor characterized by diffuse infiltration into the normal brain parenchyma. Neural stem cells are known to possess the tumor-tropic migratory capacity and thus can be used as cellular vehicles for targeted delivery of therapeutic agents. In the present study, we evaluated the efficacy of herpes simplex virus thymidine kinase (HSV-TK) suicide gene therapy for glioblastoma using neural stem/progenitor cells (NS/PCs) derived from human induced pluripotent stem cells (hiPSCs). Although transduction of hiPSCs is preferable for a safe and stable supply in the clinical setting, high-level and/or constitutive HSV-TK expression was highly cytotoxic to hiPSCs. To overcome this problem, we used the tetracycline-inducible system to control the expression of HSV-TK. hiPSC-derived NS/PCs expressing HSV-TK were transplanted in an orthotopic xenograft mouse model of human glioblastoma. Glioblastoma cell growth in mice was dramatically inhibited following ganciclovir (GCV) administration. Survival of the mice was significantly prolonged with administration of GCV compared with control groups. Time-lapse imaging of organotypic brain slice cultures first demonstrated the directional migration of NS/PCs toward glioblastoma cells and the bystander killing effect upon GCV treatment. hiPSC-derived NS/PCs with HSV-TK/GCV suicide gene system may have considerable therapeutic potential for the treatment of glioblastoma. Color images are available online.

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Overexpression of a panel of cancer stem cell markers enhances the predictive capability of the progression and recurrence in the early stage cholangiocarcinoma

Padthaisong S., Thanee M., Namwat N., Phetcharaburanin J., Klanrit P., Khuntikeo N., Titapun A., Sungkhamanon S., Saya H., Loilome W.

Journal of Translational Medicine (Journal of Translational Medicine) 18 ( 1 ) 2020.02

© 2020 The Author(s). Background: Cancer recurrence is the important problem of cholangiocarcinoma (CCA) patients, lead to a very high mortality rate. Therefore, the identification of candidate markers to predict CCA recurrence is needed in order to effectively manage the disease. This study aims to examine the predictive value of cancer stem cell (CSC) markers on the progression and recurrence of CCA patients. Methods: The expression of 6 putative CSC markers, cluster of differentiation 44 (CD44), CD44 variant 6 (CD44v6), CD44 variants 8-10 (CD44v8-10), cluster of differentiation 133 (CD133), epithelial cell adhesion molecule (EpCAM), and aldehyde dehydrogenase 1A1 (ALDH1A1), was investigated in 178 CCA tissue samples using immunohistochemistry (IHC) and analyzed with respect to clinicopathological data and patient outcome including recurrence-free survival (RFS) and overall survival (OS). The candidate CSC markers were also investigated in serum from CCA patients, and explored for their predictive ability on CCA recurrence. Results: Elevated protein level of CD44 and positive expression of CD44v6 and CD44v8-10 were significantly associated with short RFS and OS, while high levels of ALDH1A1 were correlated with a favorable prognosis patient. The elevated CD44v6 level was also correlated with higher tumor staging, whereas a decreasing level of ALDH1A1 was correlated with lower tumor staging. The levels of CD44, CD44v6 and CD44v8-10 were also correlated and were associated with a poor outcome. Furthermore, soluble CD44, CD44v6, CD44v8-10 and EpCAM were significantly increased in the recurrence group for early stage CCA; they also correlated with high levels of the tumor marker CA19-9. Elevated levels of CD44, CD44v6, CD44v8-10 or EpCAM alone or in combination has the potential to predict CCA recurrence. Conclusions: The overexpression of CD44, CD44v6, CD44v8-10 and EpCAM increases predictability of post-operative CCA recurrence. Moreover, the overexpression of the panel of CSC markers combined with CA19-9 could improve our predictive ability for tumor recurrence in early stage CCA patients. This result may be beneficial for the patients in order to predict the outcome after treatment and may be useful for clinical intervention in order to improve patient survival.

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A potent blood–brain barrier-permeable mutant IDH1 inhibitor suppresses the growth of glioblastoma with IDH1 mutation in a patient-derived orthotopic xenograft model

Machida Y., Nakagawa M., Matsunaga H., Yamaguchi M., Ogawara Y., Shima Y., Yamagata K., Katsumoto T., Hattori A., Itoh M., Seki T., Nishiya Y., Nakamura K., Suzuki K., Imaoka T., Baba D., Suzuki M., Sampetrean O., Saya H., Ichimura K., Kitabayashi I.

Molecular Cancer Therapeutics (Molecular Cancer Therapeutics) 19 ( 2 ) 375 - 383 2020.02

ISSN 15357163

©2019 American Association for Cancer Research. Gliomas are the second most common primary brain tumors in adults. They are treated with combination therapies, including surgery, radiotherapy, and chemotherapy. There are currently limited treatment options for recurrent gliomas, and new targeted therapies need to be identified, especially in glioblastomas, which have poor prognosis. Isocitrate dehydrogenase (IDH) mutations are detected in various tumors, including gliomas. Most patients with IDH mutant glioma harbor the IDH1R132H subtype. Mutant IDH catalyzes the conversion of a-ketoglutarate to the oncometabolite 2-hydroxyglutarate (2-HG), which induces aberrant epigenetic status and contributes to malignant progression, and is therefore a potential therapeutic target for IDH mutant tumors. The present study describes a novel, orally bioavailable selective mutant IDH1 inhibitor, DS-1001b. The drug has high blood–brain barrier (BBB) permeability and inhibits IDH1R132H. Continuous administration of DS-1001b impaired tumor growth and decreased 2-HG levels in subcutaneous and intracranial xenograft models derived from a patient with glioblastoma with IDH1 mutation. Moreover, the expression of glial fibrillary acidic protein was strongly induced by DS-1001b, suggesting that inhibition of mutant IDH1 promotes glial differentiation. These results reveal the efficacy of BBB-permeable DS-1001b in orthotopic patient-derived xenograft models and provide a preclinical rationale for the clinical testing of DS-1001b in recurrent gliomas.

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Identification of kit-ligand a as the gene responsible for the medaka pigment cell mutant few melanophore

Otsuki Y., Okuda Y., Naruse K., Saya H.

G3: Genes, Genomes, Genetics (G3: Genes, Genomes, Genetics) 10 ( 1 ) 311 - 319 2020.01

Copyright © 2020 Otsuki et al. The body coloration of animals is due to pigment cells derived from neural crest cells, which are multipotent and differentiate into diverse cell types. Medaka (Oryzias latipes) possesses four distinct types of pigment cells known as melanophores, xanthophores, iridophores, and leucophores. The few melanophore (fm) mutant of medaka is characterized by reduced numbers of melanophores and leucophores. We here identify kit-ligand a (kitlga) as the gene whose mutation gives rise to the fm phenotype. This identification was confirmed by generation of kitlga knockout medaka and the findings that these fish also manifest reduced numbers of melanophores and leucophores and fail to rescue the fm mutant phenotype. We also found that expression of sox5, pax7a, pax3a, and mitfa genes is down-regulated in both fm and kitlga knockout medaka, implicating c-Kit signaling in regulation of the expression of these genes as well as the encoded transcription factors in pigment cell specification. Our results may provide insight into the pathogenesis of c-Kit–related pigmentation disorders such as piebaldism in humans, and our kitlga knockout medaka may prove useful as a tool for drug screening.

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The DNA topoisomerase II inhibitor amsacrine as a novel candidate adjuvant in a model of glaucoma filtration surgery

Yamamoto K., Kokubun T., Sato K., Akaishi T., Shimazaki A., Nakamura M., Shiga Y., Tsuda S., Omodaka K., Saya H., Nakazawa T.

Scientific Reports (Scientific Reports) 9 ( 1 ) 2019.12

© 2019, The Author(s). Treatments for refractory glaucoma include trabeculectomy, in which a filtering bleb is created to reduce aqueous pressure. Mitomycin C (MMC) is often used as an adjuvant to reduce post-trabeculectomy bleb scarring and consequent failure. However, scarring sometimes still occurs. Thus, we searched for more effective trabeculectomy adjuvants with high-throughput screening (HTS) of a library of 1,165 off-patent drug compounds. This revealed that amsacrine (AMSA), a DNA topoisomerase II (TOP2) inhibitor, was the top candidate. Compared to MMC, rabbits that underwent trabeculectomy with 10% AMSA had lower IOP at 42, 56, and 70 days (P < 0.01 at all measurement points) and a higher bleb score at 28, 42, 56, and 70 days (P = < 0.01, 0.04, 0.04, and < 0.01, respectively). Compared to saline, rabbits that received 1% AMSA also had lower IOP and better bleb score at all time points, without a sharp drop in IOP just after surgery (all P < 0.01). Both effects were milder than MMC at 7 days (P = 0.02 and <0.01, respectively). Thus, this study showed that HTS may help identify new, promising uses for off-patent drugs. Furthermore, trabeculectomy with AMSA at a suitable concentration may improve the prognosis after trabeculectomy compared to MMC.

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Ropinirole hydrochloride remedy for amyotrophic lateral sclerosis – Protocol for a randomized, double-blind, placebo-controlled, single-center, and open-label continuation phase I/IIa clinical trial (ROPALS trial)

Morimoto S., Takahashi S., Fukushima K., Saya H., Suzuki N., Aoki M., Okano H., Nakahara J.

Regenerative Therapy (Regenerative Therapy) 11 143 - 166 2019.12

© 2019 The Japanese Society for Regenerative Medicine Introduction: Amyotrophic lateral sclerosis (ALS) is an intractable and incurable neurological disease. It is a progressive disease characterized by muscle atrophy and weakness caused by selective vulnerability of upper and lower motor neurons. In disease research, it has been common to use mouse models carrying mutations in responsible genes for familial ALS as pathological models of ALS. However, there is no model that has reproduced the actual conditions of human spinal cord pathology. Thus, we developed a method of producing human spinal motor neurons using human induced pluripotent stem cells (iPSCs) and an innovative experimental technique for drug screening. As a result, ropinirole hydrochloride was eventually discovered after considering such results as its preferable transitivity in the brain and tolerability, including possible adverse reactions. Therefore, we explore the safety, tolerability and efficacy of ropinirole hydrochloride as an ALS treatment in this clinical trial. Methods: The ROPALS trial is a single-center double-blind randomized parallel group-controlled trial of the safety, tolerability, and efficacy of the ropinirole hydrochloride extended-release tablet (Requip CR) at 2- to 16-mg doses in patients with ALS. Twenty patients will be recruited for the active drug group (fifteen patients) and placebo group (five patients). All patients will be able to receive the standard ALS treatment of riluzole if not changed the dosage during this trial. The primary outcome will be safety and tolerability at 24 weeks, defined from the date of randomization. Secondary outcome will be the efficacy, including any change in the ALS Functional Rating Scale-Revised (ALSFRS-R), change in the Combined Assessment of Function and Survival (CAFS), and the composite endpoint as a sum of Z-transformed scores on various clinical items. Notably, we will perform an explorative search for a drug effect evaluation using the patient-derived iPSCs to prove this trial concept. Eligible patients will have El Escorial Possible, clinically possible and laboratory-supported, clinically probable, or clinically definite amyotrophic lateral sclerosis with disease duration less than 60 months (inclusive), an ALSFRS-R score ≥2 points on all items and age from 20 to 80 years. Conclusion: Patient recruitment began in December 2018 and the last patient is expected to complete the trial protocol in November 2020. Trial registration: Current controlled trials UMIN000034954 and JMA-IIA00397 Protocol version: version 1.6 (Date; 5/Apr/2019).

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Glutaminolysis-related genes determine sensitivity to xCT-targeted therapy in head and neck squamous cell carcinoma

Okazaki S., Umene K., Yamasaki J., Suina K., Otsuki Y., Yoshikawa M., Minami Y., Masuko T., Kawaguchi S., Nakayama H., Banno K., Aoki D., Saya H., Nagano O.

Cancer Science (Cancer Science) 110 ( 11 ) 3453 - 3463 2019.11

ISSN 13479032

© 2019 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association. Targeting the function of membrane transporters in cancer stemlike cells is a potential new therapeutic approach. Cystine-glutamate antiporter xCT expressed in CD44 variant (CD44v)-expressing cancer cells contributes to the resistance to oxidative stress as well as cancer therapy through promoting glutathione (GSH)-mediated antioxidant defense. Amino acid transport by xCT might, thus, be a promising target for cancer treatment, whereas the determination factors for cancer cell sensitivity to xCT-targeted therapy remain unclear. Here, we demonstrate that high expression of xCT and glutamine transporter ASCT2 is correlated with undifferentiated status and diminished along with cell differentiation in head and neck squamous cell carcinoma (HNSCC). The cytotoxicity of the xCT inhibitor sulfasalazine relies on ASCT2-dependent glutamine uptake and glutamate dehydrogenase (GLUD)-mediated α-ketoglutarate (α-KG) production. Metabolome analysis revealed that sulfasalazine treatment triggers the increase of glutamate-derived tricarboxylic acid cycle intermediate α-KG, in addition to the decrease of cysteine and GSH content. Furthermore, ablation of GLUD markedly reduced the sulfasalazine cytotoxicity in CD44v-expressing stemlike HNSCC cells. Thus, xCT inhibition by sulfasalazine leads to the impairment of GSH synthesis and enhancement of mitochondrial metabolism, leading to reactive oxygen species (ROS) generation and, thereby, triggers oxidative damage. Our findings establish a rationale for the use of glutamine metabolism (glutaminolysis)-related genes, including ASCT2 and GLUD, as biomarkers to predict the efficacy of xCT-targeted therapy for heterogeneous HNSCC tumors.

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Neuroprotective effects of memantine via enhancement of autophagy

Hirano K., Fujimaki M., Sasazawa Y., Yamaguchi A., Ishikawa K., Miyamoto K., Souma S., Furuya N., Imamichi Y., Yamada D., Saya H., Akamatsu W., Saiki S., Hattori N.

Biochemical and Biophysical Research Communications (Biochemical and Biophysical Research Communications) 518 ( 1 ) 161 - 170 2019.10

ISSN 0006291X

© 2019 Elsevier Inc. Introduction: Chemical intervention of autophagy has been investigated in clinical trials for various age-related conditions such as sarcopenia and neurodegeneration. However, at present, no autophagy inducer has been established as a disease-modifying agent against neurodegenerative diseases. Methods: We screened a library consisting of 796 medicines clinically approved (in Japan) for autophagy enhancers as potential neurodegeneration therapeutics using HeLa cells stably expressing green fluorescent protein-microtubule-associated protein light chain 3 (GFP-LC3) followed by an analysis of the molecular mechanisms using various neuronal models. Results: The primary screening identified 152 hits in a static cellular state. A widely available Alzheimer's disease drug, memantine, which antagonizes N-Methyl-D-aspartate receptor (NMDAR), was one of the hits. Memantine increased the levels of LC3-II in a dose-dependent and time-dependent manner, and upregulated autophagic flux. In addition, the pharmacological effects of memantine on autophagy were independent of mTORC1 activity and NMDAR activation. Furthermore, a VPS34 inhibitor suppressed the memantine-induced LC3-II upregulation, suggesting that memantine may affect VPS34 complex activity. Notably, intracellular Huntington's disease-specific aggregates of elongated huntingtin, a well-established autophagy substrate, were significantly decreased by memantine. In addition, memantine enhanced elimination of degraded mitochondrial in neurons derived from induced pluripotent stem cells of PARK2 or PARK6 patients, who exhibited defective PINK1/parkin-mediated mitophagy, suggests that memantine accelerated the clearance of damaged mitochondria. Conclusion: These findings indicate that memantine may be beneficial for the treatment of neurodegeneration characterized by the abnormal accumulation of autophagy or mitophagy substrates.

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IMP dehydrogenase-2 drives aberrant nucleolar activity and promotes tumorigenesis in glioblastoma

Kofuji S., Hirayama A., Eberhardt A., Kawaguchi R., Sugiura Y., Sampetrean O., Ikeda Y., Warren M., Sakamoto N., Kitahara S., Yoshino H., Yamashita D., Sumita K., Wolfe K., Lange L., Ikeda S., Shimada H., Minami N., Malhotra A., Morioka S., Ban Y., Asano M., Flanary V., Ramkissoon A., Chow L., Kiyokawa J., Mashimo T., Lucey G., Mareninov S., Ozawa T., Onishi N., Okumura K., Terakawa J., Daikoku T., Wise-Draper T., Majd N., Kofuji K., Sasaki M., Mori M., Kanemura Y., Smith E., Anastasiou D., Wakimoto H., Holland E., Yong W., Horbinski C., Nakano I., DeBerardinis R., Bachoo R., Mischel P., Yasui W., Suematsu M., Saya H., Soga T., Grummt I., Bierhoff H., Sasaki A.

Nature cell biology (Nature cell biology) 21 ( 8 ) 1003 - 1014 2019.08

ISSN 14657392

In many cancers, high proliferation rates correlate with elevation of rRNA and tRNA levels, and nucleolar hypertrophy. However, the underlying mechanisms linking increased nucleolar transcription and tumorigenesis are only minimally understood. Here we show that IMP dehydrogenase-2 (IMPDH2), the rate-limiting enzyme for de novo guanine nucleotide biosynthesis, is overexpressed in the highly lethal brain cancer glioblastoma. This leads to increased rRNA and tRNA synthesis, stabilization of the nucleolar GTP-binding protein nucleostemin, and enlarged, malformed nucleoli. Pharmacological or genetic inactivation of IMPDH2 in glioblastoma reverses these effects and inhibits cell proliferation, whereas untransformed glia cells are unaffected by similar IMPDH2 perturbations. Impairment of IMPDH2 activity triggers nucleolar stress and growth arrest of glioblastoma cells even in the absence of functional p53. Our results reveal that upregulation of IMPDH2 is a prerequisite for the occurance of aberrant nucleolar function and increased anabolic processes in glioblastoma, which constitutes a primary event in gliomagenesis.

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ROCK inhibition induces terminal adipocyte differentiation and suppresses tumorigenesis in chemoresistant osteosarcoma cells

Takahashi N., Nobusue H., Shimizu T., Sugihara E., Yamaguchi-Iwai S., Onishi N., Kunitomi H., Kuroda T., Saya H.

Cancer Research (Cancer Research) 79 ( 12 ) 3088 - 3099 2019.06

ISSN 00085472

© 2019 American Association for Cancer Research. Tumors comprise heterogeneous cell types including cancer stem cells (CSC), progenitor cells, and differentiated cells. Chemoresistance is a potential cause of relapse and a key characteristic of CSC, but the development of novel therapeutic approaches for targeting these cells has been limited. We previously established osteosarcoma-initiating (OSi) cells by introducing the gene for c-Myc into bone marrow stromal cells of Ink4a/Arf knockout mice. These OSi cells are composed of two distinct clones: highly tumorigenic cells (AX cells), similar to bipotent committed osteochondral progenitor cells, and tripotent cells of low tumorigenicity (AO cells), similar to mesenchymal stemcells. Here we show that depolymerization of the actin cytoskeleton induces terminal adipocyte differentiation and suppresses tumorigenesis in chemoresistant OSi cells. In contrast to AX cells, AO cells were highly resistant to conventional chemotherapeutic agents such as doxorubicin and were thus identified as chemoresistant cells. Inhibition of Rho-associated coiled-coil containing protein kinase (ROCK) elicited terminal adipocyte differentiation in chemoresistant AO cells through negative regulation of the transcriptional coactivator megakaryoblastic leukemia 1 associated with actin depolymerization. The clinically administered ROCK inhibitor fasudil significantly suppressed growth in vitro and tumorigenicity in vivo of chemoresistant AO cells as well as of OSi cells. Our findings thus suggest a new therapeutic strategy based on the induction of trans-terminal differentiation via modulation of actin cytoskeleton dynamics for therapyresistant osteosarcoma stem cells.

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Glucose transporter 1-mediated vascular translocation of nanomedicines enhances accumulation and efficacy in solid tumors

Suzuki K., Miura Y., Mochida Y., Miyazaki T., Toh K., Anraku Y., Melo V., Liu X., Ishii T., Nagano O., Saya H., Cabral H., Kataoka K.

Journal of Controlled Release (Journal of Controlled Release) 301 28 - 41 2019.05

ISSN 01683659

© 2019 Elsevier B.V. Nanomedicine modification with ligands directed to receptors on tumor blood vessels has the potential for selectively enhancing nanomedicine accumulation in malignant tissues by overcoming the vascular barrier of tumors. Nevertheless, the development of broadly applicable ligand approaches capable of promoting the transvascular transport of nanomedicines in a wide spectrum of tumors has been elusive so far. By considering the indispensable and persistent glycolytic fueling of tumors, we developed glucose-installed polymeric micelles loading cisplatin (Gluc-CDDP/m) targeting the glucose transporter 1 (GLUT1), which is overexpressed in most tumors and present on vascular endothelial cells, toward improving the delivery efficiency and therapeutic efficacy. The design of the glucose ligands on Gluc-CDDP/m was engineered to control the conjugation via the carbon 6 of the glucose moieties, as well as the ligand density on the poly (ethylene glycol) (PEG) shell of the micelles. The series of micelles was then studied in vitro and in vivo against GLUT1-high human squamous cell carcinoma of the head and neck OSC-19 cells and GLUT1-low human glioblastoma-astrocytoma U87MG cells. Our results showed that precisely tuning the micelles to have glucose ligands on 25% of their PEG chains increased the efficacy against the tumors by significantly enhancing the tumor accumulation, even in GLUT1-low U87MG tumors. The enhancement of the intratumoral levels of these micelles was hindered by concomitant administration of glucose, or the GLUT1 inhibitor STF-31, confirming a GLUT1/glucose-mediated increment of the accumulation. Intravital confocal laser scanning microscopy imaging of tumor tissues further demonstrated the rapid extravasation and penetration of Gluc-CDDP/m in OSC-19 tumors compared to non-targeted CDDP/m. These findings indicate GLUT1-targeting as a promising approach for overcoming the vascular barrier and boosting the delivery of nanomedicine in tumors.

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Visualization of spatiotemporal dynamics of human glioma stem cell invasion

Tamura R., Miyoshi H., Sampetrean O., Shinozaki M., Morimoto Y., Iwasawa C., Fukaya R., Mine Y., Masuda H., Maruyama T., Narita M., Saya H., Yoshida K., Okano H., Toda M.

Molecular brain (Molecular brain) 12 ( 1 ) 2019.05

Glioblastoma exhibits phenotypic and genetic heterogeneity, aggressive invasiveness, therapeutic resistance, and tumor recurrence, which can be explained by the existence of glioma stem cells (GSCs). In this study, we visualized the spatiotemporal dynamics of invasion of human GSCs in an orthotopic xenograft mouse model using time-lapse imaging of organotypic brain slice cultures and three-dimensional imaging of optically cleared whole brains. GSCs implanted in the striatum exhibited directional migration toward axon bundles, perivascular area, and the subventricular zone around the inferior horn of the lateral ventricle. GSCs migrated in a helical pattern around axon bundles in the striatum and invaded broadly in both the rostral and caudal directions. GSCs in the corpus callosum migrated more rapidly and unidirectionally toward the contralateral side with pseudopod extension. These characteristics of GSC invasion shared histological features observed in glioblastoma patients. Spatiotemporal visualization techniques can contribute to the elucidation of the mechanisms underlying GSC invasion that may lead to the development of effective therapy for glioblastoma.

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An evolving story of the metastatic voyage of ovarian cancer cells: cellular and molecular orchestration of the adipose-rich metastatic microenvironment

Motohara T., Masuda K., Morotti M., Zheng Y., El-Sahhar S., Chong K., Wietek N., Alsaadi A., Karaminejadranjbar M., Hu Z., Artibani M., Gonzalez L., Katabuchi H., Saya H., Ahmed A.

Oncogene (Oncogene) 38 ( 16 ) 2885 - 2898 2019.04

ISSN 09509232

© 2018, The Author(s). Metastasis is a complex multistep process that involves critical interactions between cancer cells and a variety of stromal components in the tumor microenvironment, which profoundly influence the different aspects of the metastatic cascade and organ tropism of disseminating cancer cells. Ovarian cancer is the most lethal gynecological malignancy and is characterized by peritoneal disseminated metastasis. Evidence has demonstrated that ovarian cancer possesses specific metastatic tropism for the adipose-rich omentum, which has a pivotal role in the creation of the metastatic tumor microenvironment in the intraperitoneal cavity. Considering the distinct biology of ovarian cancer metastasis, the elucidation of the cellular and molecular mechanisms underlying the reciprocal interplay between ovarian cancer cells and surrounding stromal cell types in the adipose-rich metastatic microenvironment will provide further insights into the development of novel therapeutic approaches for patients with advanced ovarian cancer. Herein, we review the biological mechanisms that regulate the highly orchestrated crosstalk between ovarian cancer cells and various cancer-associated stromal cells in the metastatic tumor microenvironment with regard to the omentum by illustrating how different stromal cells concertedly contribute to the development of ovarian cancer metastasis and metastatic tropism for the omentum.

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Sulfasalazine could modulate the CD44v9-xCT system and enhance cisplatin-induced cytotoxic effects in metastatic bladder cancer

Ogihara K., Kikuchi E., Okazaki S., Hagiwara M., Takeda T., Matsumoto K., Kosaka T., Mikami S., Saya H., Oya M.

Cancer Science (Cancer Science) 110 ( 4 ) 1431 - 1441 2019.04

ISSN 13479032

© 2019 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association. The prognostic role of CD44v9, a variant isoform of CD44 and a new cell surface marker of cancer stem cells, remains unclear in bladder cancer (BC) patients. Furthermore, limited information is available on the functional role of sulfasalazine (SSZ), which could modulate the CD44v9-xCT system in order to enhance cisplatin (CDDP)-induced cytotoxicity and inhibit the metastatic potential of BC. CD44v9 protein expression was examined immunohistochemically in 63 muscle invasive BC (MIBC) patients who underwent radical cystectomy. CD44v9 expression was independently associated with disease recurrence and cancer-specific death in MIBC. Cytotoxic effects, glutathione levels, and reactive oxygen species production by SSZ and CD44v9 and phospho-p38 MAPK protein expression by SSZ with or without CDDP were assessed in MBT-2V cells with highly metastatic potential. Sulfasalazine exerted cytotoxic effects against MBT-2V cells by inhibiting glutathione levels and inducing the production of reactive oxygen species. Sulfasalazine in combination with CDDP appeared to exert strong cytotoxic effects against MBT-2V cells by inhibiting CD44v9 expression and upregulating phospho-p38 MAPK expression. The inhibitory effects of SSZ with or without CDDP were also investigated using an MBT-2V lung metastatic model. In the murine lung metastatic BC model, SSZ significantly prolonged animal survival. Furthermore, the combination of SSZ with CDDP exerted stronger inhibitory effects on the establishment of lung tumor nodules than SSZ or CDDP alone. CD44v9 expression could be a clinical biomarker for predicting poor outcomes in MIBC patients. Sulfasalazine in combination with CDDP has potential as a novel therapy against metastatic BC.

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Endothelial-mesenchymal transition drives expression of CD44 variant and xCT in pulmonary hypertension

Isobe S., Kataoka M., Endo J., Moriyama H., Okazaki S., Tsuchihashi K., Katsumata Y., Yamamoto T., Shirakawa K., Yoshida N., Shimoda M., Chiba T., Masuko T., Hakamata Y., Kobayashi E., Saya H., Fukuda K., Sano M.

American Journal of Respiratory Cell and Molecular Biology (American Journal of Respiratory Cell and Molecular Biology) 61 ( 3 ) 367 - 379 2019

ISSN 10441549

© 2019 by the American Thoracic Society. Pulmonary arterial hypertension (PAH) pathogenesis shares similarities with carcinogenesis. One CD44 variant (CD44v) isoform, CD44v8-10, binds to and stabilizes the cystine transporter subunit (xCT), producing reduced glutathione and thereby enhancing the antioxidant defense of cancer stem cells. Pharmacological inhibition of xCT by sulfasalazine suppresses tumor growth, survival, and resistance to chemotherapy. We investigated whether the CD44v-xCT axis contributes to PAH pathogenesis. CD44v was predominantly expressed on endothelial-to-mesenchymal transition (EndMT)-like cells in the neointimal layer of PAH affected pulmonary arterioles. In vitro, CD44 standard form and CD44v were induced as a result of EndMT. Among human pulmonary artery endothelial cells that have undergone EndMT, CD44v+ cells showed high levels of xCT expression on their cell surfaces and high concentrations of glutathione for survival. This made CD44v+ cells the most vulnerable target for sulfasalazine. CD44v+xCThi cells showed the highest expression levels of proinflammatory cytokines, antioxidant enzymes, antiapoptotic molecules, and cyclin-dependent kinase inhibitors. In the Sugen5416/hypoxia mouse model, CD44v+ cells were present in the thickened pulmonary vascular wall. The administration of sulfasalazine started either at the same time as "Sugen5416" administration (a prevention model) or after the development of pulmonary hypertension (a reversal model) attenuated the muscularization of the pulmonary vessels, decreased the expression of markers of inflammation, and reduced the right ventricular systolic pressure, while reducing CD44v+ cells. In conclusion, CD44v+xCThi cells appear during EndMT and in pulmonary hypertension tissues. Sulfasalazine is expected to be a novel therapeutic agent for PAH, most likely targeting EndMT-derived CD44v+xCThi cells.

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Cancer Stem-Cell Marker CD44v9-Positive Cells Arise From Helicobacter pylori–Infected CAPZA1-Overexpressing Cells

Tsugawa H., Kato C., Mori H., Matsuzaki J., Kameyama K., Saya H., Hatakeyama M., Suematsu M., Suzuki H.

Cellular and Molecular Gastroenterology and Hepatology (Cellular and Molecular Gastroenterology and Hepatology) 8 ( 3 ) 319 - 334 2019

© 2019 The Authors Background & Aims: CD44 variant 9 (CD44v9)-positive cancer stem-like cells strongly contribute to the development and recurrence of gastric cancer. However, the origin of CD44v9-positive cells is uncertain. Methods: CD44v9, β-catenin, and epithelial splicing regulatory protein 1 signals were assessed by real-time reverse-transcription polymerase chain reaction, immunoblot analysis, or immunofluorescence microscopy. Capping actin protein of muscle Z-line α subunit 1 (CAPZA1) expression was assessed by immunoblot analysis or immunohistochemical analysis of Mongolian gerbils' gastric mucosa or human biopsy specimens. Levels of oxidative stress were assessed by measuring malondialdehyde and protein carbonylation. Histone H3 acetylation levels in the CAPZA1 proximal promoter region were measured by using chromatin immunoprecipitation analysis with an antibody against the acetylated histone H3 in human gastric carcinoma cell line (AGS) cells. Results: CD44v9 is expressed in CAPZA1-overexpressing cells in human gastric cancer tissues. CAPZA1 overexpression enhanced expression of β-catenin, which is a transcription factor for CD44, and epithelial splicing regulatory protein 1, which increases alternative splicing of CD44 to generate CD44v9. CAPZA1-overexpressing cells after cytotoxin-associated gene A accumulation showed CD44v9 expression by inducing nuclear accumulation of β-catenin, concomitant with the enhancement of expression of Sal-like protein 4 and Krüppel-like factor 5, which encode reprogramming factors. Oxidative stress increased the CAPZA1 expression in AGS cells through the enhancement of histone H3 acetylation of CAPZA1 promoter. CAPZA1 expression was increased depending on oxidative stress in H pylori–infected gastric mucosa. Conclusions: CD44v9 expression is evoked from CAPZA1-overexpressing cells after accumulation of cytotoxin-associated gene A. Our findings provide important insights into the mechanisms underlying the development of CD44v9-positive cells.

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Vasodilator oxyfedrine inhibits aldehyde metabolism and thereby sensitizes cancer cells to xCT-targeted therapy

Otsuki Y., Yamasaki J., Suina K., Okazaki S., Koike N., Saya H., Nagano O.

Cancer Science (Cancer Science) 111 ( 1 ) 127 - 136 2019

ISSN 13479032

© 2019 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association. The major cellular antioxidant glutathione (GSH) protects cancer cells from oxidative damage that can lead to the induction of ferroptosis, an iron-dependent form of cell death triggered by the aberrant accumulation of lipid peroxides. Inhibitors of the cystine-glutamate antiporter subunit xCT, which mediates the uptake of extracellular cystine and thereby promotes GSH synthesis, are thus potential anticancer agents. However, the efficacy of xCT-targeted therapy has been found to be diminished by metabolic reprogramming that affects redox status in cancer cells. Identification of drugs for combination with xCT inhibitors that are able to overcome resistance to xCT-targeted therapy might thus provide the basis for effective cancer treatment. We have now identified the vasodilator oxyfedrine (OXY) as a sensitizer of cancer cells to GSH-depleting agents including the xCT inhibitor sulfasalazine (SSZ). Oxyfedrine contains a structural motif required for covalent inhibition of aldehyde dehydrogenase (ALDH) enzymes, and combined treatment with OXY and SSZ was found to induce accumulation of the cytotoxic aldehyde 4-hydroxynonenal and cell death in SSZ-resistant cancer cells both in vitro and in vivo. Microarray analysis of tumor xenograft tissue showed cyclooxygenase-2 expression as a potential biomarker for the efficacy of such combination therapy. Furthermore, OXY-mediated ALDH inhibition was found to sensitize cancer cells to GSH depletion induced by radiation therapy in vitro. Our findings thus establish a rationale for repurposing of OXY as a sensitizing drug for cancer treatment with agents that induce GSH depletion.

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CD44 variant inhibits insulin secretion in pancreatic β cells by attenuating LAT1-mediated amino acid uptake

Kobayashi Nana, Okazaki Shogo, Sampetrean Oltea, Irie Junichiro, Itoh Hiroshi, Saya Hideyuki

Scientific Reports (Scientific Reports) 8 ( 1 ) 2018.12

ISSN 2045-2322

CD44 variant (CD44v) contributes to cancer stemness by stabilizing the xCT subunit of system xc(-) and thereby promoting its glutamate-cystine antiporter activity. CD44 has also been implicated in autoimmune insulitis and inflammation in diabetic islets, but whether CD44v regulates insulin secretion has remained unclear. Here we show that CD44v inhibits insulin secretion by attenuating amino acid transport mediated by the L-type amino acid transporter LAT1. CD44v expression level was inversely related to insulin content in islets of normal and diabetic model mice. Knockdown of CD44 increased insulin secretion, the intracellular insulin level, and the transport of neutral amino acids mediated by LAT1 in Min6 cells. Attenuation of the uptake of neutral amino acids with a LAT inhibitor reduced insulin secretion and insulin content in Min6 cells, whereas overexpression of LAT1 increased insulin secretion. Moreover, inhibition of LAT1 prevented the increase in insulin secretion and content induced by CD44 depletion in Min6 cells. Our results thus implicate CD44v in the regulation of insulin secretion and reveal that amino acid transport is rate limiting for such secretion. They further suggest that amino acid transport mediated by LAT1 is a potential therapeutic target for diabetes.

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Gold-nanofève surface-enhanced Raman spectroscopy visualizes hypotaurine as a robust anti-oxidant consumed in cancer survival

Shiota Megumi, Naya Masayuki, Yamamoto Takehiro, Hishiki Takako, Tani Takeharu, Takahashi Hiroyuki, Kubo Akiko, Koike Daisuke, Itoh Mai, Ohmura Mitsuyo, Kabe Yasuaki, Sugiura Yuki, Hiraoka Nobuyoshi, Morikawa Takayuki, Takubo Keiyo, Suina Kentaro, Nagashima Hideaki, Sampetrean Oltea, Nagano Osamu, Saya Hideyuki, Yamazoe Shogo, Watanabe Hiroyuki, Suematsu Makoto

Nature Communications (Nature Communications) 9 ( 1 ) 2018.12

ISSN 2041-1723

Gold deposition with diagonal angle towards boehmite-based nanostructure creates random arrays of horse-bean-shaped nanostructures named gold-nanofève (GNF). GNF generates many electromagnetic hotspots as surface-enhanced Raman spectroscopy (SERS) excitation sources, and enables large-area visualization of molecular vibration fingerprints of metabolites in human cancer xenografts in livers of immunodeficient mice with sufficient sensitivity and uniformity. Differential screening of GNF-SERS signals in tumours and those in parenchyma demarcated tumour boundaries in liver tissues. Furthermore, GNF-SERS combined with quantum chemical calculation identified cysteine-derived glutathione and hypotaurine (HT) as tumour-dominant and parenchyma-dominant metabolites, respectively. CD44 knockdown in cancer diminished glutathione, but not HT in tumours. Mechanisms whereby tumours sustained HT under CD44-knockdown conditions include upregulation of PHGDH, PSAT1 and PSPH that drove glycolysis-dependent activation of serine/glycine-cleavage systems to provide one-methyl group for HT synthesis. HT was rapidly converted into taurine in cancer cells, suggesting that HT is a robust anti-oxidant for their survival under glutathione-suppressed conditions.

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Tranilast inhibits the expression of genes related to epithelial-mesenchymal transition and angiogenesis in neurofibromin-deficient cells

Harigai Ritsuko, Sakai Shigeki, Nobusue Hiroyuki, Hirose Chikako, Sampetrean Oltea, Minami Noriaki, Hata Yukie, Kasama Takashi, Hirose Takanori, Takenouchi Toshiki, Kosaki Kenjiro, Kishi Kazuo, Saya Hideyuki, Arima Yoshimi

Scientific Reports (Scientific Reports) 8 ( 1 ) 2018.12

ISSN 2045-2322

Neurofibromatosis type 1 (NF1) is caused by germline mutations in the NF1 gene and is characterized by café au lait spots and benign tumours known as neurofibromas. NF1 encodes the tumour suppressor protein neurofibromin, which negatively regulates the small GTPase Ras, with the constitutive activation of Ras signalling resulting from NF1 mutations being thought to underlie neurofibroma development. We previously showed that knockdown of neurofibromin triggers epithelial-mesenchymal transition (EMT) signalling and that such signalling is activated in NF1-associated neurofibromas. With the use of a cell-based drug screening assay, we have now identified the antiallergy drug tranilast (N-(3,4-dimethoxycinnamoyl) anthranilic acid) as an inhibitor of EMT and found that it attenuated the expression of mesenchymal markers and angiogenesis-related genes in NF1-mutated sNF96.2 cells and in neurofibroma cells from NF1 patients. Tranilast also suppressed the proliferation of neurofibromin-deficient cells in vitro more effectively than it did that of intact cells. In addition, tranilast inhibited sNF96.2 cell migration and proliferation in vivo. Knockdown of type III collagen (COL3A1) also suppressed the proliferation of neurofibroma cells, whereas expression of COL3A1 and SOX2 was increased in tranilast-resistant cells, suggesting that COL3A1 and the transcription factor SOX2 might contribute to the development of tranilast resistance.

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Epidermal growth factor receptor promotes glioma progression by regulating xCT and GluN2B-containing N-methyl-d-aspartate–sensitive glutamate receptor signaling

Suina K., Tsuchihashi K., Yamasaki J., Kamenori S., Shintani S., Hirata Y., Okazaki S., Sampetrean O., Baba E., Akashi K., Mitsuishi Y., Takahashi F., Takahashi K., Saya H., Nagano O.

Cancer Science (Cancer Science) 109 ( 12 ) 3874 - 3882 2018.12

ISSN 13479032

© 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association. Autocrine and paracrine factors, including glutamate and epidermal growth factor (EGF), are potent inducers of brain tumor cell invasion, a pathological hallmark of malignant gliomas. System xc(–) consists of xCT and CD98hc subunits and functions as a plasma membrane antiporter for the uptake of extracellular cystine in exchange for intracellular glutamate. We previously showed that the EGF receptor (EGFR) interacts with xCT and thereby promotes the activity of system xc(–) in a kinase-independent manner, resulting in enhanced glutamate release in glioma cells. However, the molecular mechanism underlying EGFR-mediated glioma progression in a glutamate-rich microenvironment has remained unclear. Here we show that the GluN2B subunit of the N-methyl-d-aspartate–sensitive glutamate receptor (NMDAR) is a substrate of EGFR in glioma cells. In response to EGF stimulation, EGFR phosphorylated the COOH-terminal domain of GluN2B and thereby enhanced glutamate-NMDAR signaling and consequent cell migration in EGFR-overexpressing glioma cells. Treatment with the NMDAR inhibitor MK-801 or the system xc(–) inhibitor sulfasalazine suppressed EGF-elicited glioma cell migration. The administration of sulfasalazine and MK-801 also synergistically suppressed the growth of subcutaneous tumors formed by EGFR-overexpressing glioma cells. Furthermore, shRNA-mediated knockdown of xCT and GluN2B cooperatively prolonged the survival of mice injected intracerebrally with such glioma cells. Our findings thus establish a central role for EGFR in the signaling crosstalk between xCT and GluN2B-containing NMDAR in glioma cells.

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T-type Calcium Channels Determine the Vulnerability of Dopaminergic Neurons to Mitochondrial Stress in Familial Parkinson Disease

Tabata Y., Imaizumi Y., Sugawara M., Andoh-Noda T., Banno S., Chai M., Sone T., Yamazaki K., Ito M., Tsukahara K., Saya H., Hattori N., Kohyama J., Okano H.

Stem Cell Reports (Stem Cell Reports) 11 ( 5 ) 1171 - 1184 2018.11

ISSN 22136711

© 2018 The Author(s) Parkinson disease (PD) is a progressive neurological disease caused by selective degeneration of dopaminergic (DA) neurons in the substantia nigra. Although most cases of PD are sporadic cases, familial PD provides a versatile research model for basic mechanistic insights into the pathogenesis of PD. In this study, we generated DA neurons from PARK2 patient-specific, isogenic PARK2 null and PARK6 patient-specific induced pluripotent stem cells and found that these neurons exhibited more apoptosis and greater susceptibility to rotenone-induced mitochondrial stress. From phenotypic screening with an FDA-approved drug library, one voltage-gated calcium channel antagonist, benidipine, was found to suppress rotenone-induced apoptosis. Furthermore, we demonstrated the dysregulation of calcium homeostasis and increased susceptibility to rotenone-induced stress in PD, which is prevented by T-type calcium channel knockdown or antagonists. These findings suggest that calcium homeostasis in DA neurons might be a useful target for developing new drugs for PD patients. Our study demonstrate the dysregulation of calcium homeostasis and increased susceptibility to rotenone-induced stress in PD patient-derived DA neurons, which are further prevented by T-type calcium channel antagonists. These findings suggest that calcium homeostasis in DA neurons would be a useful target for developing new drugs for PD patients.

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Inhibiting xCT improves 5-fluorouracil resistance of gastric cancer induced by CD44 variant 9 expression

Miyoshi S., Tsugawa H., Matsuzaki J., Hirata K., Mori H., Saya H., Kanai T., Suzuki H.

Anticancer Research (Anticancer Research) 38 ( 11 ) 6163 - 6170 2018.11

ISSN 02507005

© 2018 International Institute of Anticancer Research.All right reserved. Background/Aim: Cancer stem cells (CSCs) play a critical role in resistance to chemotherapy. CD44 is a cell surface marker of CSCs. CD44 variant 9 (CD44v9) interacts with a cystine-glutamate antiporter (xCT) and is an unfavorable predictive factor in gastric cancer. We investigated the impact of CD44v9 expression on 5-fluorouracil (5-FU) resistance and the efficacy of the xCT inhibitor, sulfasalazine (SASP), in improving drug resistance. Materials and Methods: The human gastric cancer cell line MKN28 was transfected with pRc/CMV plasmids encoding human CD44 or CD44v9, which were used for in vitro and in vivo experiments. Results: CD44v9 expression results in 5-FU resistance by increasing intracellular glutathione and suppressing the drug-induced production of reactive oxygen species (ROS). SASP improved the drug sensitivity of CD44v9-expressing cells. Conclusion: Inhibition of xCT improved the clinical efficacy of chemotherapy against gastric cancer. CD44v9 expression can be a novel biomarker to predict resistance against 5-FU in gastric cancer.

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Modeling sporadic ALS in iPSC-derived motor neurons identifies a potential therapeutic agent

Fujimori K., Ishikawa M., Otomo A., Atsuta N., Nakamura R., Akiyama T., Hadano S., Aoki M., Saya H., Sobue G., Okano H.

Nature Medicine (Nature Medicine) 24 ( 10 ) 1579 - 1589 2018.10

ISSN 10788956

© 2018, The Author(s). Amyotrophic lateral sclerosis (ALS) is a heterogeneous motor neuron disease for which no effective treatment is available, despite decades of research into SOD1-mutant familial ALS (FALS). The majority of ALS patients have no familial history, making the modeling of sporadic ALS (SALS) essential to the development of ALS therapeutics. However, as mutations underlying ALS pathogenesis have not yet been identified, it remains difficult to establish useful models of SALS. Using induced pluripotent stem cell (iPSC) technology to generate stem and differentiated cells retaining the patients’ full genetic information, we have established a large number of in vitro cellular models of SALS. These models showed phenotypic differences in their pattern of neuronal degeneration, types of abnormal protein aggregates, cell death mechanisms, and onset and progression of these phenotypes in vitro among cases. We therefore developed a system for case clustering capable of subdividing these heterogeneous SALS models by their in vitro characteristics. We further evaluated multiple-phenotype rescue of these subclassified SALS models using agents selected from non-SOD1 FALS models, and identified ropinirole as a potential therapeutic candidate. Integration of the datasets acquired in this study permitted the visualization of molecular pathologies shared across a wide range of SALS models.

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Therapeutic potential of tranilast for the treatment of chronic graft-versus-host disease in mice

Mukai S., Ogawa Y., Saya H., Kawakami Y., Tsubota K.

PLoS ONE (PLoS ONE) 13 ( 10 ) 2018.10

© 2018 Mukai et al.This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Chronic graft-versus-host disease (cGVHD) is a marked complication of hematopoietic stem cell transplantation, and multiple organs can be affected by cGVHD-induced inflammation and fibrosis. In clinical settings, immunosuppressive agents have been the last resort to treat cGVHD. However, it has been only partially effective for cGVHD. Hence, efficacious treatment of cGVHD is eagerly awaited. Our previous work suggested that oxidative stress was elevated in cGVHD-disordered lacrimal glands and that epithelial-To-mesenchymal transition (EMT) was implicated in fibrosis caused by ocular cGVHD. In addition, our recent article demonstrated that thioredoxin interaction protein (TXNIP) and transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) were associated with the development of cGVHD. After our search for effective drugs, we chose tranilast to combat systemic cGVHD. Tranilast is known to (1) act as an inhibitor of the inflammatory molecules TXNIP and NF-κB and (2) exert anti-fibrotic, anti-EMT and anti-oxidative effects. To investigate the effectiveness of tranilast for cGVHD, we used an MHC-compatible, multiple minor histocompatibility antigen-mismatched murine model of cGVHD. Tranilast or a solvent-vehicle were orally given to the allogeneic bone marrow transplantation (allo-BMT) recipients from the day before allo-BMT (Day-1) to Day 27 after allo-BMT. Their cGVHD-vulnerable organs were collected Day 28 after allo-BMT and analyzed by using various methods such as histology, immunohistochemistry and immunoblotting. As indicated by our results, tranilast alleviated cGVHD-elicited inflammation and fibrosis by suppressing the expression and/or activation of TXNIP and NF-κB and preventing EMT. Taken together, although this strategy may not be a complete cure for cGVHD, tranilast could be a promising medication to ameliorate cGVHD-Triggered disabling symptoms.

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Synthetic lethality of the ALDH3A1 inhibitor dyclonine and xCT inhibitors in glutathione deficiency-resistant cancer cells

Okazaki S., Shintani S., Hirata Y., Suina K., Semba T., Yamasaki J., Umene K., Ishikawa M., Saya H., Nagano O.

Oncotarget (Oncotarget) 9 ( 73 ) 33832 - 33843 2018.09

© Okazaki et al. The cystine-glutamate antiporter subunit xCT suppresses iron-dependent oxidative cell death (ferroptosis) and is therefore a promising target for cancer treatment. Given that cancer cells often show resistance to xCT inhibition resulting in glutathione (GSH) deficiency, however, we here performed a synthetic lethal screen of a drug library to identify agents that sensitize the GSH deficiency-resistant cancer cells to the xCT inhibitor sulfasalazine. This screen identified the oral anesthetic dyclonine which has been recently reported to act as a covalent inhibitor for aldehyde dehydrogenases (ALDHs). Treatment with dyclonine induced intracellular accumulation of the toxic aldehyde 4-hydroxynonenal in a cooperative manner with sulfasalazine. Sulfasalazine-resistant head and neck squamous cell carcinoma (HNSCC) cells were found to highly express ALDH3A1 and knockdown of ALDH3A1 rendered these cells sensitive to sulfasalazine. The combination of dyclonine and sulfasalazine cooperatively suppressed the growth of highly ALDH3A1-expressing HNSCC or gastric tumors that were resistant to sulfasalazine monotherapy. Our findings establish a rationale for application of dyclonine as a sensitizer to xCT-targeted cancer therapy.

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HER2 heterogeneity is associated with poor survival in HER2-positive breast cancer

Hosonaga Mari, Arima Yoshimi, Sampetrean Oltea, Komura Daisuke, Koya Ikuko, Sasaki Takashi, Sato Eiichi, Okano Hideyuki, Kudoh Jun, Ishikawa Shumpei, Saya Hideyuki, Ishikawa Takashi

International journal of molecular sciences (International Journal of Molecular Sciences) 19 ( 8 ) 2018.08

ISSN 1661-6596

Intratumoral human epidermal growth factor receptor 2 (HER2) heterogeneity has been reported in 16–36% of HER2-positive breast cancer and its clinical impact is under discussion. We examined the biological effects of HER2-heterogeneity on mouse models and analyzed metastatic brains by RNA sequence analysis. A metastatic mouse model was developed using 231-Luc (triple negative cells) and 2 HER2-positive cell lines, namely, HER2-60 and HER2-90 which showed heterogeneous and monotonous HER2 expressions, respectively. Metastatic lesions developed in 3 weeks in all the mice injected with HER2-60 cells, and in 69% of the mice injected with HER2-90 and 87.5% of the mice injected with 231-Luc. The median survival days of mice injected with 231-Luc, HER2-60, and HER2-90 cells were 29 (n = 24), 24 (n = 22) and 30 (n = 13) days, respectively. RNA sequence analysis showed that CASP-1 and its related genes were significantly downregulated in metastatic brain tumors with HER2-60 cells. The low expression of caspase-1 could be a new prognostic biomarker for early relapse in HER2-positive breast cancer.

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Development of a functional thyroid model based on an organoid culture system

Saito Yoshiyuki, Onishi Nobuyuki, Takami Hiroshi, Seishima Ryo, Inoue Hiroyoshi, Hirata Yuki, Kameyama Kaori, Tsuchihashi Kenji, Sugihara Eiji, Uchino Shinya, Ito Koichi, Kawakubo Hirofumi, Takeuchi Hiroya, Kitagawa Yuko, Saya Hideyuki, Nagano Osamu

Biochemical and Biophysical Research Communications (Biochemical and Biophysical Research Communications) 497 ( 2 ) 783 - 789 2018.03

ISSN 0006-291X

The low turnover rate of thyroid follicular cells and the lack of a long-term thyroid cell culture system have hampered studies of thyroid carcinogenesis. We have now established a thyroid organoid culture system that supports thyroid cell proliferation in vitro. The established mouse thyroid organoids performed thyroid functions including thyroglobulin synthesis, iodide uptake, and the production and release of thyroid hormone. Furthermore, transplantation of the organoids into recipient mice resulted in the formation of normal thyroid–like tissue capable of iodide uptake and thyroglobulin production in vivo. Finally, forced expression of oncogenic NRAS (NRAS^Q61R) in thyroid organoids established from p53 knockout mice and transplantation of the manipulated organoids into mouse recipients generated a model of poorly differentiated thyroid cancer. Our findings suggest that this newly developed thyroid organoid culture system is a potential research tool for the study of thyroid physiology and pathology including thyroid cancer.

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Prospects for new lung cancer treatments that target EMT signaling

Otsuki Yuji, Saya Hideyuki, Arima Yoshimi

Developmental Dynamics (Developmental Dynamics) 247 ( 3 ) 462 - 472 2018.03

ISSN 1058-8388

Lung cancer is the most common cancer worldwide. Treatment options for lung cancer include surgery, radiation therapy, chemotherapy, molecularly targeted therapy including epidermal growth factor receptor or anaplastic lymphoma kinase inhibitors, and immunotherapy. These treatments can be administered alone or in combination. Despite therapeutic advances, however, lung cancer remains the leading cause of cancer death. Recent studies have indicated that epithelial-mesenchymal transition (EMT) is associated with malignancy in various types of cancer, and activation of EMT signaling in cancer cells is widely considered to contribute to metastasis, recurrence, or therapeutic resistance. In this review, we provide an overview of the role of EMT in the progression of lung cancer. We also discuss the prospects for new therapeutic strategies that target EMT signaling in lung cancer. Developmental Dynamics 247:462–472, 2018.

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Metabolic heterogeneity and plasticity of glioma stem cells in a mouse glioblastoma model

Shibao Shunsuke, Minami Noriaki, Koike Naoyoshi, Fukui Nobuyuki, Yoshida Kazunari, Saya Hideyuki, Sampetrean Oltea

Neuro-Oncology (Neuro-Oncology) 20 ( 3 ) 343 - 354 2018.02

ISSN 1522-8517

Background. Glioblastomas have been shown to rely on glycolysis as an energy source. However, recent evidence suggests that at least a subset of glioma cells with stem cell-like properties can thrive on oxidative phosphorylation. It remains unclear whether both metabolic phenotypes support tumor propagation, if they are independent, and how stable they are. The present study investigated these questions with the use of isogenic murine glioma stem cells (GSCs). Methods. GSCs were established from tumors formed by Ink4a/Arf-null, H-RasV12-expressing glioma-initiating cells that differed in extracellular acidification potential. Metabolic characteristics of GSCs were determined by measurement of glucose, oxygen, and glutamine uptake, ATP content, and lactate production. Effects of metabolic inhibitors and changes in oxygen or nutrient availability on lactate production and tumorsphere growth were also determined. Results. GSCs were found either to consume more glucose and produce more lactate or to consume more oxygen and maintain a higher ATP content depending on the metabolic characteristics of the tumor cells of origin. The latter, mitochondrial-type GSCs increased lactate production after treatment with the oxidative phosphorylation inhibitor oligomycin or phenformin. Exposure to hypoxia also increased lactate production and expression of glycolysis-related enzymes and metabolites in mitochondrial-type GSCs in a reversible manner. Conclusions. Both glycolytic and mitochondrial-type energy production can sustain tumor propagation by isogenic GSCs. Whereas both phenotypes can be independent and stable, cells that rely on oxidative phosphorylation can also switch to a more glycolytic phenotype in response to metabolic stress, suggesting that plasticity is a further characteristic of GSC metabolism.

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Recurrent Spindle Cell Carcinoma Shows Features of Mesenchymal Stem Cells

Ouchi T., Morikawa S., Shibata S., Takahashi M., Yoshikawa M., Soma T., Miyashita H., Muraoka W., Kameyama K., Kawana H., Arima Y., Saya H., Okano H., Nakagawa T., Asoda S.

Journal of Dental Research (Journal of Dental Research) 97 ( 7 ) 779 - 786 2018.02

ISSN 0022-0345

This study investigated a case of spindle cell carcinoma (SpCC) in tongue pathological lesions. The patient experienced a local recurrence and distant metastasis after surgical intervention. Although standard chemotherapy was administered, a granulomatous mass continued to develop. This aggressive growth led to survival of the tumor. Secondary debulking surgery was performed to improve the patient’s quality of life at the request of the patient. Using a tissue sample derived from the secondary debulking surgery, we performed an analysis of the tumor’s cell surface antigens, differentiation potential, metastatic ability, and inhibition potential by anticancer reagents. In vitro analysis revealed that the cell population grown under adherent culture conditions expressed the mesenchymal stem cell (MSC) markers CD73, CD90, and CD105. The cell line established from this SpCC contained colony-forming unit fibroblasts (CFU-Fs) and exhibited multipotent differentiation into several mesenchymal lineages, including bone, cartilage, and fat. The SpCC cells also displayed vigorous mobilization. These characteristics suggested that they had the differentiation potential of mesenchymal cells, especially MSCs, rather than that of epithelial cells. The surgical specimen analyzed in this study resisted the molecular target reagent cetuximab, which is an epidermal growth factor receptor inhibitor. This clinical insight revealed that chemotherapy-resistant SpCC cells have different characteristics compared to most other cancer cells, which are sensitive to cetuximab. Our cell death assay revealed that SpCC cell death was induced by the anticancer drug imatinib, which is known to inhibit protein tyrosine kinase activity of ABL, platelet-derived growth factor receptor α (PDGFRα), and KIT. Here, we report recurrent SpCC with characteristics of MSCs and potential for treatment with imatinib.

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Variant isoforms of CD44 involves acquisition of chemoresistance to cisplatin and has potential as a novel indicator for identifying a cisplatin-resistant population in urothelial cancer

Hagiwara Masayuki, Kikuchi Eiji, Tanaka Nobuyuki, Kosaka Takeo, Mikami Shuji, Saya Hideyuki, Oya Mototsugu

BMC Cancer (BMC Cancer) 18 ( 1 ) 2018.01

ISSN 1471-2407

Background: Cisplatin is the most commonly used chemotherapeutic agent in the treatment of patients with metastatic and/or recurrent urothelial cancer. However, the effectiveness of these treatments is severely limited due to the development of cisplatin resistance. Cancer stem cells have been documented as one of the key hypotheses involved in chemoresistance. CD44v8-10 has been identified as one of the new cancer stem cells markers and was recently shown to enhance the antioxidant system by interaction with xCT, a subunit of the cystine transporter modulating intracellular glutathione synthesis. The aim of the present study was to investigate the clinical role of CD44v8-10 and the molecular mechanism underlying the acquisition of cisplatin resistance through CD44v8-10 in urothelial cancer. Methods: We analyzed the clinical significance of the immunohistochemical CD44v9 expression, which detects the immunogen of human CD44v8-10, in 77 urothelial cancer patients treated with cisplatin-based systemic chemotherapy for recurrence and/or metastasis. We then evaluated the biological role of CD44v8-10 in the acquisition of cisplatin resistance using the urothelial cancer cell lines, T24 and T24PR, which were generated to acquire resistance to cisplatin. Results: The 5-year cancer-specific survival rate was significantly lower in the CD44v9-positive group than in the CD44v9-negative group (P = 0.008). Multivariate analyses revealed that CD44v9 positivity was an independent risk factor of cancer-specific survival (P = 0.024, hazard ratio = 5.16) in urothelial cancer patients who had recurrence and/or metastasis and received cisplatin-based chemotherapy. The expression of CD44v8-10 and xCT was stronger in T24PR cells than in T24 cells. The amount of intracellular glutathione was significantly higher in T24PR cells than in T24 cells (p < 0.001), and intracellular reactive oxygen species production by cisplatin was lower in T24PR cells than in T24 cells. Furthermore, the knockdown of CD44v8-10 by siRNA led to the recovery of cisplatin sensitivity in T24PR cells. Conclusions: CD44v9 in tumor specimens has potential as a novel indicator for identifying a cisplatin-chemoresistant population among urothelial cancer patients. CD44v8-10 contributes to reactive oxygen species defenses, which are involved in chemoresistance, by promoting the function of xCT, which adjusts the synthesis of glutathione.

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Periostin antisense oligonucleotide suppresses bleomycin-induced formation of a lung premetastatic niche for melanoma

Semba Takashi, Sugihara Eiji, Kamoshita Nagisa, Ueno Sayaka, Fukuda Keitaro, Yoshino Masafumi, Takao Kazumasa, Yoshikawa Kazunori, Izuhara Kenji, Arima Yoshimi, Suzuki Makoto, Saya Hideyuki

Cancer Science (Cancer Science) 109 ( 5 ) 1447 - 1454 2018.01

ISSN 1347-9032

Metastasis is the leading cause of cancer death. A tumor-supportive microenvironment, or premetastatic niche, at potential secondary tumor sites plays an important role in metastasis, especially in tumor cell colonization. Although a fibrotic milieu is known to promote tumorigenesis and metastasis, the underlying molecular contributors to this effect have remained unclear. Here we show that periostin, a component of the extracellular matrix that functions in tissue remodeling, has a key role in formation of a fibrotic environment that promotes tumor metastatic colonization. We found that periostin was widely expressed in fibrotic lesions of mice with bleomycin-induced lung fibrosis, and that up-regulation of periostin expression coincided with activation of myofibroblasts positive for α-smooth muscle actin. We established a lung metastasis model for B16 murine melanoma cells and showed that metastatic colonization of the lung by these cells was markedly promoted by bleomycin-induced lung fibrosis. Inhibition of periostin expression by giving an intratracheal antisense oligonucleotide targeting periostin mRNA was found to suppress bleomycin-induced lung fibrosis and thereby to attenuate metastatic colonization of the lung by melanoma cells. Our results indicate that periostin is a key player in the development of bleomycin-induced fibrosis and consequent enhancement of tumor cell colonization in the lung. Our results therefore implicate periostin as a potential target for prevention or treatment of lung metastasis.

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Dynamic changes in CD44v-positive cells after preoperative anti-HER2 therapy and its correlation with pathologic complete response in HER2-positive breast cancer

Yamauchi Teruo, Fernandez Jose Rodrigo Espinosa, Imamura Chiyo K., Yamauchi Hideko, Jinno Hiromitsu, Takahashi Maiko, Kitagawa Yuko, Nakamura Seigo, Lim Bora, Krishnamurthy Savitri, Reuben James M., Liu Diane, Tripathy Debasish, Chen Helen, Takebe Naoko, Saya Hideyuki, Ueno Naoto T.

Oncotarget (Oncotarget) 9 ( 6 ) 6872 - 6882 2018.01

ISSN 1949-2553

Chemotherapy has been reported to increase the proportion of cancer stem cells (CSCs) and to promote epithelial-mesenchymal transition (EMT) phenotype changes. Anti-HER2 therapy may provide a strategy for eliminating CSC and EMT, which contribute to therapeutic resistance. No study has determined the changes in the quantity or characteristics of CSCs or circulating tumor cells (CTCs) with EMT phenotype during preoperative anti-HER2 therapy, and whether these changes correlate to response to dual anti-HER2 therapy. In a prospective clinical trial to evaluate pharmacodynamic biomarkers, 18 patients with operable primary HER2-positive breast cancer received dual anti-Her2 preoperative therapy with trastuzumab and lapatinib with paclitaxel. Proportions of tumor cells with CSC characteristics and EMT markers in CTC's were estimated at baseline, after 6 and 18 weeks of preoperative therapy to determine the quantitative cutoff value to predict pathologic complete response (pCR). Out of 18 patients, 8 (44%) had a pCR; 5 of these 8 patients (62%) were positive for CD44v at baseline and none were positive on the 6-week biopsy. In contrast, 6 of the 10 patients without pCR exhibited persistent levels, or enrichment of CD44v proportion and expression at 6 and 18 weeks (p=0.0128). Other biomarkers were not statistically significant predictors of pCR. Enrichment of CD44v-positive tumor cells after dual anti-HER2 therapy alone may predict poor response to dual anti-HER2 therapy plus chemotherapy.

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Modeling phenotypes of malignant gliomas

Sampetrean Oltea, Saya Hideyuki

Cancer Science (Cancer Science) 109 ( 1 ) 6 - 14 2018.01

ISSN 1347-9032

Malignant gliomas are primary tumors of the central nervous system characterized by diffuse infiltration into the brain and a high recurrence rate. Advances in comprehensive genomic studies have provided unprecedented insight into the genetic and molecular heterogeneity of these tumors and refined our understanding of their evolution from low to high grade. However, similar levels of phenotypic characterization are indispensable to understanding the complexity of malignant gliomas. Experimental glioma models have also achieved great progress in recent years. Advances in transgenic technologies and cell culture have allowed the establishment of mouse models that mirror the human disease with increasing fidelity and which support single-cell resolution for phenotypic analyses. Here we review the major types of preclinical glioma models, with an emphasis on how recent developments in experimental modeling have shed new light on two fundamental aspects of glioma phenotype, their cell of origin and their invasive potential.

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Stat3 as a potential therapeutic target for rheumatoid arthritis

Oike Takatsugu, Sato Yuiko, Kobayashi Tami, Miyamoto Kana, Nakamura Satoshi, Kaneko Yosuke, Kobayashi Shu, Harato Kengo, Saya Hideyuki, Matsumoto Morio, Nakamura Masaya, Niki Yasuo, Miyamoto Takeshi

Scientific Reports 7 ( 1 ) 2017.12

ISSN 2045-2322

Rheumatoid arthritis (RA) is a multi-factorial disease characterized by chronic inflammation and destruction of multiple joints. To date, various biologic treatments for RA such as anti-Tumor necrosis factor alpha antibodies have been developed; however, mechanisms underlying RA development remain unclear and targeted therapy for this condition has not been established. Here, we provide evidence that signal transducer and activator of transcription 3 (Stat3) promotes inflammation and joint erosion in a mouse model of arthritis. Stat3 global KO mice show early embryonic lethality; thus, we generated viable Stat3 conditional knockout adult mice and found that they were significantly resistant to collagen-induced arthritis (CIA), the most common RA model, compared with controls. We then used an in vitro culture system to screen ninety-six existing drugs to select Stat3 inhibitors and selected five candidate inhibitors. Among them, three significantly inhibited development of arthritis and joint erosion in CIA wild-Type mice. These findings suggest that Stat3 inhibitors may serve as promising drugs for RA therapy.

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Dual blockade of the lipid kinase PIP4Ks and mitotic pathways leads to cancer-selective lethality

Kitagawa Mayumi, Liao Pei Ju, Lee Kyung Hee, Wong Jasmine, Shang See Cheng, Minami Noriaki, Sampetrean Oltea, Saya Hideyuki, Lingyun Dai, Prabhu Nayana, Diam Go Ka, Sobota Radoslaw, Larsson Andreas, Nordlund Pär, McCormick Frank, Ghosh Sujoy, Epstein David M., Dymock Brian W., Lee Sang Hyun

Nature Communications 8 ( 1 ) 2017.12

ISSN 2041-1723

Achieving robust cancer-specific lethality is the ultimate clinical goal. Here, we identify a compound with dual-inhibitory properties, named a131, that selectively kills cancer cells, while protecting normal cells. Through an unbiased CETSA screen, we identify the PIP4K lipid kinases as the target of a131. Ablation of the PIP4Ks generates a phenocopy of the pharmacological effects of PIP4K inhibition by a131. Notably, PIP4Ks inhibition by a131 causes reversible growth arrest in normal cells by transcriptionally upregulating PIK3IP1, a suppressor of the PI3K/Akt/mTOR pathway. Strikingly, Ras activation overrides a131-induced PIK3IP1 upregulation and activates the PI3K/Akt/mTOR pathway. Consequently, Ras-transformed cells override a131-induced growth arrest and enter mitosis where a131's ability to de-cluster supernumerary centrosomes in cancer cells eliminates Ras-activated cells through mitotic catastrophe. Our discovery of drugs with a dual-inhibitory mechanism provides a unique pharmacological strategy against cancer and evidence of cross-activation between the Ras/Raf/MEK/ERK and PI3K/AKT/mTOR pathways via a RasPI3K signaling network.

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Reducing inflammatory cytokine production from renal collecting duct cells by inhibiting GATA2 ameliorates acute kidney injury

Yu Lei, Moriguchi Takashi, Kaneko Hiroshi, Hayashi Makiko, Hasegawa Atsushi, Nezu Masahiro, Saya Hideyuki, Yamamoto Masayuki, Shimizu Ritsuko

Molecular and Cellular Biology 37 ( 22 ) 2017.11

ISSN 0270-7306

Acute kidney injury (AKI) is a leading cause of chronic kidney disease. Proximal tubules are considered to be the primary origin of pathogenic inflammatory cytokines in AKI. However, it remains unclear whether other cell types, including collecting duct (CD) cells, participate in inflammatory processes. The transcription factor GATA2 is specifically expressed in CD cells and maintains their cellular identity. To explore the pathophysiological function of GATA2 in AKI, we generated renal tubular cell-specific Gata2 deletion (G2CKO) mice and examined their susceptibility to ischemia reperfusion injury (IRI). Notably, G2CKO mice exhibited less severe kidney damage, with reduced granulomacrophagic infiltration upon IRI. Transcriptome analysis revealed that a series of inflammatory cytokine genes were downregulated in GATA2-deficient CD cells, suggesting that GATA2 induces inflammatory cytokine expression in diseased kidney CD cells. Through high-throughput chemical library screening, we identified a potent GATA inhibitor. The chemical reduces cytokine production in CD cells and protects the mouse kidney from IRI. These results revealed a novel pathological mechanism of renal IRI, namely, that CD cells produce inflammatory cytokines and promote IRI progression. In injured kidney CD cells, GATA2 exerts a proinflammatory function by upregulating inflammatory cytokine gene expression. GATA2 can therefore be considered a therapeutic target for AKI.

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CD44 variant–dependent regulation of redox balance in EGFR mutation–positive non–small cell lung cancer

Kawano Yuko, Iwama Eiji, Tsuchihashi Kenji, Shibahara Daisuke, Harada Taishi, Tanaka Kentaro, Nagano Osamu, Saya Hideyuki, Nakanishi Yoichi, Okamoto Isamu

Lung Cancer 113 72 - 78 2017.11

ISSN 0169-5002

Objectives The regulation of redox balance in cancer cells is an important factor in tumor development and chemoresistance, with oncogene activation having been shown to induce the generation of reactive oxygen species (ROS). Activating mutations of the epidermal growth factor receptor gene (EGFR) are oncogenic drivers in non–small cell lung cancer (NSCLC), but it has remained unknown whether ligand-independent EGFR signaling conferred by EGFR mutation triggers ROS generation in NSCLC cells. Materials and Methods HEK293T cells were transfected with an expression vector for mutant EGFR. The expression of CD44 variant (CD44v) isoforms in NSCLC cell lines was evaluated by flow cytometry. Cells were depleted of CD44v by RNA interference and assayed for ROS and glutathione (GSH) levels. The effect of CD44v on cisplatin sensitivity was evaluated in vitro with the MTS assay. Results EGFR signaling due to EGFR mutation increased ROS levels in transfected HEK293T cells. The expression of CD44v isoforms was found to be inversely correlated with basal ROS levels in EGFR mutation–positive NSCLC cell lines. Knockdown of CD44v induced depletion of intracellular GSH and increased ROS levels in EGFR-mutated NSCLC cells that express CD44v at a high level (CD44v^high). In addition, depletion of GSH by treatment with buthionine-[S, R]-sulfoximine induced marked accumulation of ROS and enhanced the cytotoxicity of cisplatin in CD44v^high EGFR-mutated NSCLC cells but not in corresponding CD44v^low cells. This enhancement of cisplatin cytotoxicity by GSH depletion was prevented by treatment with the antioxidant N-acetyl-L-cysteine. Knockdown of CD44v also enhanced cisplatin cytotoxicity in CD44v^high EGFR mutation–positive NSCLC cells but not in CD44v^low cells. Conclusion Our results thus implicate CD44v in redox adaptation and as a potential target for treatment in CD44v^high EGFR-mutated NSCLC cells.

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Organotypic brain explant culture as a drug evaluation system for malignant brain tumors

Minami Noriaki, Maeda Yusuke, Shibao Shunsuke, Arima Yoshimi, Ohka Fumiharu, Kondo Yutaka, Maruyama Koji, Kusuhara Masatoshi, Sasayama Takashi, Kohmura Eiji, Saya Hideyuki, Sampetrean Oltea

Cancer Medicine 6 ( 11 ) 2635 - 2645 2017.11

Therapeutic options for malignant brain tumors are limited, with new drugs being continuously evaluated. Organotypic brain slice culture has been adopted for neuroscience studies as a system that preserves brain architecture, cellular function, and the vascular network. However, the suitability of brain explants for anticancer drug evaluation has been unclear. We here adopted a mouse model of malignant glioma based on expression of H-Ras^V12 in Ink4a/Arf^−/− neural stem/progenitor cells to establish tumor-bearing brain explants from adult mice. We treated the slices with cisplatin, temozolomide, paclitaxel, or tranilast and investigated the minimal assays required to assess drug effects. Serial fluorescence-based tumor imaging was sufficient for evaluation of cisplatin, a drug with a pronounced cytotoxic action, whereas immunostaining of cleaved caspase 3 (a marker of apoptosis) and of Ki67 (a marker of cell proliferation) was necessary for the assessment of temozolomide action and immunostaining for phosphorylated histone H3 (a marker of mitosis) allowed visualization of paclitaxel-specific effects. Staining for cleaved caspase 3 was also informative in the assessment of drug toxicity for normal brain tissue. Incubation of explants with fluorescently labeled antibodies to CD31 allowed real-time imaging of the microvascular network and complemented time-lapse imaging of tumor cell invasion into surrounding tissue. Our results suggest that a combination of fluorescence imaging and immunohistological staining allows a unified assessment of the effects of various classes of drug on the survival, proliferation, and invasion of glioma cells, and that organotypic brain slice culture is therefore a useful tool for evaluation of antiglioma drugs.

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Establishment of pten knockout medaka with transcription activator–like effector nucleases (TALENs) as a model of PTEN deficiency disease

Matsuzaki Yuriko, Sakuma Tetsushi, Yamamoto Takashi, Saya Hideyuki

PLoS One 12 ( 10 ) 2017.10

ISSN 1932-6203

Phosphatase and tensin homolog (PTEN) is a lipid and protein phosphatase that antagonizes signaling by the phosphatidylinositol 3-kinase (PI3K)–AKT signaling pathway. The PTEN gene is a major tumor suppressor, with mutations of this gene occurring frequently in tumors of humans and mice. We have now developed mutant medaka deficient in PTEN with the use of transcription activator–like effector nuclease (TALEN) technology. Medaka possesses two pten genes, ptena and ptenb, similar to zebrafish. We established 16 ptena mutant lines and two ptenb mutant lines. Homozygous single pten mutants were found to be viable and fertile. In contrast, pten double-knockout (dko) embryos manifested severe abnormalities in vasculogenesis, eye size, and tail development at 72 hours post fertilization (hpf) and died before hatching. Immunoblot analysis revealed that the ratio of phosphorylated to total forms of AKT (pAKT/AKT) in pten dko embryos was four times that in wild-type embryos, indicative of up-regulation of signaling by the PI3K-AKT pathway. Treatment of pten dko embryos with the PI3K inhibitor LY294002 reduced the pAKT/AKT ratio by about one-half and partially rescued the defect in vasculogenesis. Additional inhibitors of the PI3K-AKT pathway, including rapamycin and N-α-tosyl-L-phenylalanyl chloromethyl ketone, also partially restored vasculogenesis in the dko embryos. Our model system thus allows pten dko embryos to be readily distinguished from wild-type embryos at an early stage of development and is suitable for the screening of drugs able to compensate for PTEN deficiency.

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Convection-enhanced delivery of sulfasalazine prolongs survival in a glioma stem cell brain tumor model

Haryu Shinya, Saito Ryuta, Jia Wenting, Shoji Takuhiro, Mano Yui, Sato Aya, Kanamori Masayuki, Sonoda Yukihiko, Sampetrean Oltea, Saya Hideyuki, Tominaga Teiji

Journal of Neuro-Oncology (Journal of Neuro-Oncology) 136 ( 1 ) 1 - 9 2017.09

ISSN 0167-594X

Expression of CD44 in glioma cells was previously correlated with tumor grade and is considered a stem cell marker. CD44 stabilizes the cystine–glutamate transporter (xCT) and inhibits apoptosis in cancer stem cells (CSCs). Recently it was found that Sulfasalazine (SSZ), an anti-inflammatory drug, acts as an inhibitor of xCT and therefore has potential as a targeted therapy for CSCs. In this study, we tested an efficacy of SSZ against glioma stem cell model developed in rats. As poor penetration of blood–brain barrier resulted in insufficient efficacy of systemic SSZ treatment, SSZ was delivered locally with convection-enhanced delivery (CED). In vitro, expression of CD44 in glioma cells and efficacy of SSZ against glioma cells and glioma stem cells were confirmed. SSZ demonstrated anti-proliferative activity in a dose dependent manner against these cells. This activity was partially reversible with the addition of antioxidant, N-acetyl-l-cysteine, to the medium. In vivo, CED successfully delivered SSZ into the rat brain parenchyma. When delivered at 5 mM concentration, which was the highest possible concentration when SSZ was dissolved in water, CED of SSZ resulted in almost no tissue damage. Against highly malignant bRiTs-G3 brain tumor xenografted rat model; the glioma stem cell model, CED of SSZ at 5 mM concentration induced apoptosis and prolonged survival. Consequently, CED of SSZ induced glioma stem cell death without evidence of tissue damage to normal brain parenchyma. This strategy may be a promising targeted treatment against glioma stem cells.

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cRGD peptide installation on cisplatin-loaded nanomedicines enhances efficacy against locally advanced head and neck squamous cell carcinoma bearing cancer stem-like cells

Miyano Kazuki, Cabral Horacio, Miura Yutaka, Matsumoto Yu, Mochida Yuki, Kinoh Hiroaki, Iwata Caname, Nagano Osamu, Saya Hideyuki, Nishiyama Nobuhiro, Kataoka Kazunori, Yamasoba Tatsuya

Journal of Controlled Release 261 275 - 286 2017.09

ISSN 0168-3659

Recalcitrant head and neck squamous cell carcinoma (HNSCC) usually relapses after therapy due to the enrichment of drug resistant cancer stem-like cells (CSCs). Nanomedicines have shown potential for eradicating both cancer cells and CSCs by effective intratumoral navigation for reaching particular cell populations and controlling drug delivery. The installation of ligands on nanomedicines is an attractive approach for improving the delivery to CSCs within tumors, though the development of CSC-selective ligand-receptor systems has been challenging. Herein, we found that the CSC subpopulation in HNSCC cells overexpresses α_vβ₅ integrins, which is preferentially expressed in tumor neovasculature and cancer cells, and can be effectively targeted by using cyclic Arg-Gly-Asp (cRGD) peptide. Thus, in this study, we propose installing cRGD peptide on micellar nanomedicines incorporating cisplatin for improving their activity against CSCs and enhancing survival. Both cisplatin-loaded micelles (CDDP/m) and cRGD-installed CDDP/m (cRGD-CDDP/m) were effective against HNSCC SAS-L1-Luc cells in vitro, though cRGD-installed CDDP/m was more potent than CDDP/m against the CSC fraction. In vivo, the cRGD-CDDP/m also showed significant antitumor activity against HNSCC orthotopic tumors, i.e. SAS-L1 and HSC-2. Moreover, cRGD-CDDP/m rapidly accumulated into the lymph node metastasis of SAS-L1 tumors, effectively inhibiting their growth, and prolonging mice survival. These findings indicate cRGD-installed nanomedicines as an advantageous strategy for targeting CSCs in HNSCC, and particularly, cRGD-CDDP/m as a significant therapeutic strategy against regionally advanced HNSCC.

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Endothelium-induced three-dimensional invasion of heterogeneous glioma initiating cells in a microfluidic coculture platform

Chonan Yuta, Taki Sotaro, Sampetrean Oltea, Saya Hideyuki, Sudo Ryo

Integrative Biology (United Kingdom) 9 ( 9 ) 762 - 773 2017.09

ISSN 1757-9694

Glioblastoma (GBM) is a highly invasive primary brain tumor that displays cellular heterogeneity, which is composed of glioma initiating cells (GICs) and their differentiated progeny. GICs play an important role in driving aggressive invasion. In particular, the interaction between GICs and blood vessels is critical because blood vessels are known to serve as routes for the invasion of GICs. However, the effect of endothelial cells on the three-dimensional (3D) invasion process of GICs as well as the spatial relationship between GICs and their differentiated progeny remains unclear. Here, we utilized a microfluidic device to recapitulate the 3D brain tumor microenvironments constituted by human umbilical vein endothelial cells (HUVECs) and type I collagen. Using the device, we found that HUVECs promoted the 3D invasion of heterogeneous glioma cell populations into type I collagen gel. The invasion induced by HUVECs was predominantly preceded by cells positive for nestin, a neural stem cell marker. In contrast, cells positive for tubulin β3 (TUBB3), a differentiated cell marker, rarely preceded invasion. In addition, HUVECs induced the upregulation of TUBB3 in GICs. Finally, we found that the genes associated with invasion, such as integrins α2 and β3, were significantly upregulated in the presence of HUVECs. These results as well as the experimental approach provide valuable knowledge for the development of effective therapeutic strategies targeting the aggressive invasion of GBM.

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ZEB1 expression is a potential indicator of invasive endometriosis

Furuya Masataka, Masuda Hirotaka, Hara Kanako, Uchida Hiroshi, Sato Kenji, Sato Suguru, Asada Hironori, Maruyama Tetsuo, Yoshimura Yasunori, Katabuchi Hidetaka, Tanaka Mamoru, Saya Hideyuki

Acta Obstetricia et Gynecologica Scandinavica 96 ( 9 ) 1128 - 1135 2017.09

ISSN 0001-6349

Introduction: Although endometriosis is a benign disease, it shares some features with cancers, such as invasiveness and the potential to metastasize. This study sought to investigate the epithelial-mesenchymal transition status in human endometriotic lesions. Material and methods: Thirteen endometriosis patients and 10 control women without endometriosis undergoing surgery for benign indications were recruited. We examined the expression of E-cadherin, vimentin, and epithelial-mesenchymal transition-induced transcriptional factors, such as Snail and ZEB1, by immunohistochemistry. We evaluated the expression of each marker in epithelial cells of both endometriotic lesions (ovarian endometrioma, deep infiltrating endometriosis, adenomyosis) and normal endometria. The correlation between ZEB1 expression and serum level of CA125 was also investigated. Results: Immunohistochemical analysis revealed that although E-cadherin, vimentin, and Snail were expressed in epithelia of normal endometria and endometriotic lesions, ZEB1 expression was only expressed in epithelia of endometriotic lesions. Additionally, ZEB1 was most frequently observed in epithelial cells of invasive endometriosis. The endometriosis patients with high serum CA125 level were more likely to have ZEB1-positive lesions. Conclusions: This is the first observation of ZEB1 expression in epithelial cells of benign disease. The preferential expression of ZEB1 in epithelial cells of endometriotic lesions suggests that these cells may have, at least in part, a higher level of mesenchymal features possibly via ZEB1-driven epithelial-mesenchymal transition than normal endometria and that ZEB1 can be a potential indicator of invasiveness or severity of endometriosis.

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Calcitriol exerts an anti-tumor effect in osteosarcoma by inducing the endoplasmic reticulum stress response

Shimizu Takatsune, Kamel Walied A., Yamaguchi-Iwai Sayaka, Fukuchi Yumi, Muto Akihiro, Saya Hideyuki

Cancer Science 108 ( 9 ) 1793 - 1802 2017.09

ISSN 1347-9032

Osteosarcoma is the most common type of primary bone tumor, and novel therapeutic approaches for this disease are urgently required. To identify effective agents, we screened a panel of Food and Drug Administration (FDA)-approved drugs in AXT cells, our newly established mouse osteosarcoma line, and identified calcitriol as a candidate compound with therapeutic efficacy for this disease. Calcitriol inhibited cell proliferation in AXT cells by blocking cell cycle progression. From a mechanistic standpoint, calcitriol induced endoplasmic reticulum (ER) stress, which was potentially responsible for downregulation of cyclin D1, activation of p38 MAPK, and intracellular production of reactive oxygen species (ROS). Knockdown of Atf4 or Ddit3 restored cell viability after calcitriol treatment, indicating that the ER stress response was indeed responsible for the anti-proliferative effect in AXT cells. Notably, the ER stress response was induced to a lesser extent in human osteosarcoma than in AXT cells, consistent with the weaker suppressive effect on cell growth in the human cells. Thus, the magnitude of ER stress induced by calcitriol might be an index of its anti-osteosarcoma effect. Although mice treated with calcitriol exhibited weight loss and elevated serum calcium levels, a single dose was sufficient to decrease osteosarcoma tumor size in vivo. Our findings suggest that calcitriol holds therapeutic potential for treatment of osteosarcoma, assuming that techniques to diminish its toxicity could be established. In addition, our results show that calcitriol could still be safely administered to osteosarcoma patients for its original purposes, including treatment of osteoporosis.

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Activation of Transforming Growth Factor Beta 1 Signaling in Gastric Cancer-associated Fibroblasts Increases Their Motility, via Expression of Rhomboid 5 Homolog 2, and Ability to Induce Invasiveness of Gastric Cancer Cells

Ishimoto Takatsugu, Miyake Keisuke, Nandi Tannistha, Yashiro Masakazu, Onishi Nobuyuki, Huang Kie Kyon, Lin Suling Joyce, Kalpana Ramnarayanan, Tay Su Ting, Suzuki Yuka, Cho Byoung Chul, Kuroda Daisuke, Arima Kota, Izumi Daisuke, Iwatsuki Masaaki, Baba Yoshifumi, Oki Eiji, Watanabe Masayuki, Saya Hideyuki, Hirakawa Kosei, Baba Hideo, Tan Patrick

Gastroenterology 153 ( 1 ) 191 - 204.e16 2017.07

ISSN 0016-5085

Background & Aims Fibroblasts that interact with cancer cells are called cancer-associated fibroblasts (CAFs), which promote progression of different tumor types. We investigated the characteristics and functions of CAFs in diffuse-type gastric cancers (DGCs) by analyzing features of their genome and gene expression patterns. Methods We isolated CAFs and adjacent non-cancer fibroblasts (NFs) from 110 gastric cancer (GC) tissues from patients who underwent gastrectomy in Japan from 2008 through 2016. Cells were identified using specific markers of various cell types by immunoblot and flow cytometry. We selected pairs of CAFs and NFs for whole-exome and RNA sequencing analyses, and compared expression of specific genes using quantitative reverse transcription PCR. Protein levels and phosphorylation were compared by immunoblot and immunofluorescence analyses. Rhomboid 5 homolog 2 (RHBDF2) was overexpressed from a transgene in fibroblasts or knocked down using small interfering RNAs. Motility and invasiveness of isolated fibroblasts and GC cell lines (AGS, KATOIII, MKN45, NUGC3, NUGC4, OCUM-2MD3 and OCUM-12 cell lines) were quantified by real-time imaging analyses. We analyzed 7 independent sets of DNA microarray data from patients with GC and associated expression levels of specific genes with patient survival times. Nude mice were given injections of OCUM-2MD3 in the stomach wall; tumors and metastases were collected and analyzed by immunohistochemistry. Results Many of the genes with increased expression in CAFs compared with NFs were associated with transforming growth factor beta 1 (TGFB1) activity. When CAFs were cultured in extracellular matrix, they became more motile than NFs; DGC cells incubated with CAFs were also more motile and invasive in vitro than DGC cells not incubated with CAFs. When injected into nude mice, CAF-incubated DGC cells invaded a greater number of lymphatic vessels than NF-incubated DGC cells. We identified RHBDF2 as a gene overexpressed in CAFs compared with NFs. Knockdown of RHBDF2 in CAFs reduced their elongation and motility in response to TGFB1, whereas overexpression of RHBDF2 in NFs increased their motility in extracellular matrix. RHBDF2 appeared to regulate oncogenic and non-canonical TGFB1 signaling. Knockdown of RHBDF2 in CAFs reduced cleavage of the TGFB receptor 1 (TGFBR1) by ADAM metallopeptidase domain 17 (ADAM17 or TACE) and reduced expression of genes that regulate motility. Incubation of NFs with in interleukin 1 alpha (IL1A), IL1B or tumor necrosis factor, secreted by DGCs, increased fibroblast expression of RHBDF2. Simultaneous high expression of these cytokines in GC samples was associated with shorter survival times of patients. Conclusions In CAFs isolated from human DGCs, we observed increased expression of RHBDF2, which regulates TGFB1 signaling. Expression of RHBDF2 in fibroblasts is induced by inflammatory cytokines (such as IL1A, IL1B, and tumor necrosis factor) secreted by DGCs. RHBDF2 promotes cleavage of TGFBR1 by activating TACE and motility of CAFs in response to TGFB1. These highly motile CAFs induce DGCs to invade extracellular matrix and lymphatic vessels in nude mice.

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Inflammatory mediator ultra-low-molecular-weight hyaluronan triggers necrosis of B-precursor leukemia cells with high surface CD44 expression

Kasai Shin, Furuichi Yoshiyuki, Ando Norie, Kagami Keiko, Abe Masako, Nakane Takaya, Goi Kumiko, Inukai Takeshi, Saitoh Sei, Ohno Shinichi, Okazaki Shogo, Nagano Osamu, Saya Hideyuki, Sugita Kanji

Cell death & disease 8 ( 6 ) e2857 2017.06

Acute lymphoblastic leukemia (ALL) with mixed lineage leukemia (MLL) gene rearrangements (MLL+ALL) has a dismal prognosis and is characterized by high surface CD44 expression. Known that CD44 has the specific binding sites for a natural ligand hyaluronan (HA), we investigated biological effects of HA with different molecular sizes on MLL+ALL cell lines, and found that the addition of ultra-low-molecular-weight (ULMW)-HA strongly suppressed their thymidine uptakes. The MLL+ALL cell line lacking surface CD44 expression established by genome editing showed no suppression of thymidine uptake. Surface CD44-high B-precursor ALL cell lines other than MLL+, but not T-ALL cell lines, were also suppressed in their thymidine uptakes. The inhibition of thymidine uptakes was because of induction of cell death, but dead cells lacked features of apoptosis on cytospin smears and flow cytometric analysis. The cell death was neither blocked by pan-caspase inhibitor nor autophagy inhibitor, but was completely blocked by necrosis inhibitor necrostatin-1. Necrotic cell death was further supported by a marked release of a high-mobility protein group B1 and morphological changes on transmission electron microscopy. Elevation of intracellular reactive oxygen species production suggested a role for inducing this necrotic cell death. ULMW-HA-triggered cell death was similarly demonstrated in surface CD44-high primary B-precursor leukemia cells. Assuming that ULMW-HA is abundantly secreted at the site of infection and inflammation, this study sheds light on understanding the mechanism of a transient inflammation-associated remission of leukemia. Further, the CD44-targeting may become an effective approach in future for the treatment of refractory B-precursor ALL by its capability of predominantly eradicating CD44-high leukemia-initiating cells.

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Variant CD44 expression is enriching for a cell population with cancer stem cell-like characteristics in human lung adenocarcinoma

Nishino Makoto, Ozaki Mari, Hegab Ahmed E., Hamamoto Junko, Kagawa Shizuko, Arai Daisuke, Yasuda Hiroyuki, Naoki Katsuhiko, Soejima Kenzo, Saya Hideyuki, Betsuyaku Tomoko

Journal of Cancer 8 ( 10 ) 1774 - 1785 2017.06

ISSN 1837-9664

Background: Preliminary studies have identified cancer stem cells (CSCs) in various cancers and there are several ongoing clinical studies targeting these cells. CD44 (standard or variant isoforms) and/or aldehyde dehydrogenase (ALDH) expression is the most commonly used markers for the identification of CSCs. The goal of the current study was to examine the ability of CD44v, either alone or in combination with ALDH, to identify CSCs within human lung cancer cells lines. Methods: We examined several lung adenocarcinoma cell lines for the ability of CD44v and/or ALDH expression to enrich for cells with CSC characteristics such as in vitro differential proliferation rate, chemotherapeutic-resistance, tumorsphere formation, and in vivo tumorigenicity. We also compared their in vivo secondary tumor formation, and histological characteristics of their xenograft tumors, and examined their expression of PD-L1, EGFR, xCT, and reactive oxygen species (ROS). Results: Both CD44v^high/ALDH^high and CD44v^high/ALDH^low cells were enriched in cells with CSC characteristics, with the CD44v^high/ALDH^low cells being more proliferative and more resistant to chemotherapeutics, whereas CD44v^high/ALDH^high cells were more efficient in forming tumorspheres in vitro, in making primary xenograft tumors, and in propagating secondary tumors in vivo. Applying stricter sorting gates to select for cells with the highest CD44v/ALDH expression caused the CD44v^high/ALDH^low cells to lose their high proliferation rates and chemotherapeutic resistance ability, but enriched for the tumorsphere-forming cells among the CD44v^high/ALDH^high and CD44v^high/ALDH^low cells. CD44v^high expression was associated with PD-L1 and xCT expression in both H1650 and HCC827 cells. This association was not modified by ALDH expression in the H1650 cell line. However, in the HCC827 cell line, ALDH expression was negatively associated with PD-L1 and positively associated with xCT expression. Conclusion: Lung adenocarcinoma cells with high CD44v expression are enriched for CSCs. Addition of ALDH as an enrichment marker bestowed some CSCs characteristics to CD44v^high/ALDH^low cells and others to CD44v^high/ALDH^high cells. We propose that lung adenocarcinoma contains different CSCs, each of them endowed with different CSC characteristics.

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Functional analysis of the DEPDC1 oncoantigen in malignant glioma and brain tumor initiating cells

Kikuchi Ryogo, Sampetrean Oltea, Saya Hideyuki, Yoshida Kazunari, Toda Masahiro

Journal of Neuro-Oncology 1 - 11 2017.05

ISSN 0167-594X

DEP domain containing 1 (DEPDC1) is a novel oncoantigen expressed in cancer cells, which presents oncogenic activity and high immunogenicity. Although DEPDC1 has been predicted to be a useful antigen for the development of a cancer vaccine, its pathophysiological roles in glioma have not been investigated. Here, we analyzed the expression and function of DEPDC1 in malignant glioma. DEPDC1 expression in glioma cell lines, glioma tissues, and brain tumor initiating cells (BTICs) was assessed by western blot and quantitative polymerase chain reaction (PCR). The effect of DEPDC1 downregulation on cell growth and nuclear factor kappa B (NFκB) signaling in glioma cells was investigated. Overall survival was assessed in mouse glioma models using human glioma cells and induced mouse brain tumor stem cells (imBTSCs) to determine the effect of DEPDC1 suppression in vivo. DEPDC1 expression was increased in glioma cell lines, tissues, and BTICs. Suppression of endogenous DEPDC1 expression by small interfering RNA (siRNA) inhibited glioma cell viability and induced apoptosis through NFκB signaling. In mouse glioma models using human glioma cells and imBTSCs, downregulation of DEPDC1 expression prolonged overall survival. These results suggest that DEPDC1 represents a target molecule for the treatment of glioma.

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Phase 1 study of sulfasalazine and cisplatin for patients with CD44v-positive gastric cancer refractory to cisplatin (EPOC1407)

Shitara Kohei, Doi Toshihiko, Nagano Osamu, Fukutani Miki, Hasegawa Hiromi, Nomura Shogo, Sato Akihiro, Kuwata Takeshi, Asai Kai, Einaga Yasuaki, Tsuchihashi Kenji, Suina Kentaro, Maeda Yusuke, Saya Hideyuki, Ohtsu Atsushi

Gastric Cancer 1 - 6 2017.05

ISSN 1436-3291

A previous dose-escalation study of sulfasalazine (SSZ), an inhibitor of cystine-glutamate exchange transporter xc (–), in the variant form of CD44 (CD44v)-positive cancer stem cells (CSCs) suggested that administration of SSZ induces the reduction of CD44v-positive cells and intracellular reduced glutathione (GSH) levels in patients with advanced gastric cancer (AGC). Here we report a study to evaluate SSZ in combination with cisplatin in patients with CD44v-expressing AGC refractory to cisplatin. SSZ was given by oral administration four times daily with 2 weeks on and 1 week off. Cisplatin at 60 mg/m² was administered every 3 weeks. Of the 15 patients who underwent prescreening of CD44v expression, 8 patients were positive, and 7 patients were treated with the dose level of SSZ at 6 g/day. One patient experienced dose-limiting toxicity (DLT) as grade 3 anorexia. Although no other patients experienced DLT, 4 patients required dose interruption or reduction of SSZ; thus, we terminated further dose escalation. No patient achieved objective response, but 1 patient completed six cycles with stable disease for more than 4 months as well as reduction of intratumoral GSH level. The combination of SSZ plus cisplatin was manageable, although dose modification was frequently required during a short observational period.

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Rheostatic CD44 isoform expression and its association with oxidative stress in human malignant mesothelioma

Chew Shan Hwu, Okazaki Yasumasa, Akatsuka Shinya, Wang Shenqi, Jiang Li, Ohara Yuuki, Ito Fumiya, Saya Hideyuki, Sekido Yoshitaka, Toyokuni Shinya

Free Radical Biology and Medicine 106 91 - 99 2017.05

ISSN 0891-5849

CD44 exists as a standard (CD44s) isoform and different variant isoforms (CD44v) due to alternative splicing. While the complex nature of these different isoforms has not been fully elucidated, CD44v expression has been shown to exert oncogenic effects by promoting tumor progression, metastasis and resistance of tumor cells to chemotherapy. One of the CD44v isoforms, CD44v8-10, was recently shown to protect cancer cells from oxidative stress by increasing the synthesis of glutathione (GSH). However, data regarding CD44 isoform expression in malignant mesothelioma (MM) are still lacking. Here, we show that most of the MM cell lines express both the CD44s and CD44v isoforms, in contrast to non-tumorigenic mesothelial cells, which express only CD44s. Moreover, we show here that these MM cell lines are positive for CD44 variable exon 9, with CD44v8-10 among the variant isoforms expressed. The expression of CD44 variable exon 9 was found to be statistically associated with NF2 inactivation, a common occurrence in MM. Knockdown of CD44 reduced the protein level of xCT, a cystine transporter, and increased oxidative stress. However, an increase in GSH was also observed and was associated with enhanced chemoresistance in CD44-knockdown cells. Increased GSH was mediated by the Nrf2/AP-1-induced upregulation of GCLC, a subunit of the enzyme catalyzing GSH synthesis. Our results thus suggest that the response to CD44 depletion is cell type-dependent and, in cases such as MM cells, compensatory pathway(s) might be activated rheostatically to account for the loss of CD44 and counteract enhanced oxidative stress.

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FZR1 loss increases sensitivity to DNA damage and consequently promotes murine and human B-cell acute leukemia

Ishizawa Jo, Sugihara Eiji, Kuninaka Shinji, Mogushi Kaoru, Kojima Kensuke, Benton Christopher B., Zhao Ran, Chachad Dhruv, Hashimoto Norisato, Jacamo Rodrigo O., Qiu Yihua, Yoo Suk Young, Okamoto Shinichiro, Andreeff Michael, Kornblau Steven M., Saya Hideyuki

Blood 129 ( 14 ) 1958 - 1968 2017.04

ISSN 0006-4971

FZR1 (fizzy-related protein homolog; also known as CDH1 [cell division cycle 20 related 1]) functions in the cell cycle as a specific activator of anaphase-promoting complex or cyclosome ubiquitin ligase, regulating late mitosis, G₁ phase, and activation of the G₂-M checkpoint. FZR1 has been implicated as both a tumor suppressor and oncoprotein, and its precise contribution to carcinogenesis remains unclear. Here, we examined the role of FZR1 in tumorigenesis and cancer therapy by analyzing tumor models and patient specimens. In an Fzr1 gene-trap mouse model of B-cell acute lymphoblastic leukemia (B-ALL), mice with Fzr1-deficient B-ALL survived longer than those with Fzr1-intact disease, and sensitivity of Fzr1-deficient B-ALL cells to DNA damage appeared increased. Consistently, conditional knockdown of FZR1 sensitized human B-ALL cell lines to DNA damage–induced cell death. Moreover, multivariate analyses of reverse-phase protein array of B-ALL specimens from newly diagnosed B-ALL patients determined that a low FZR1 protein expression level was an independent predictor of a longer remission duration. The clinical benefit of a low FZR1 expression level at diagnosis was no longer apparent in patients with relapsed B-ALL. Consistent with this result, secondary and tertiary mouse recipients of Fzr1-deficient B-ALL cells developed more progressive and radiation-resistant disease than those receiving Fzr1-intact B-ALL cells, indicating that prolonged inactivation of Fzr1 promotes the development of resistant clones. Our results suggest that reduction of FZR1 increases therapeutic sensitivity of B-ALL and that transient rather than tonic inhibition of FZR1 may be a therapeutic strategy.

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Blocking COX-2 induces apoptosis and inhibits cell proliferation via the Akt/survivin- and Akt/ID3 pathway in low-grade-glioma

Sato Aya, Mizobuchi Yoshifumi, Nakajima Kohei, Shono Kenji, Fujihara Toshitaka, Kageji Teruyoshi, Kitazato Keiko, Matsuzaki Kazuhito, Mure Hideo, Kuwayama Kazuyuki, Sumi Akiko, Saya Hideyuki, Sampetrean Oltea, Nagahirao Shinji

Journal of Neuro-Oncology 132 ( 2 ) 231 - 238 2017.04

ISSN 0167-594X

Approximately half of surgically-treated patients with low-grade-glioma (LGG) suffer recurrence or metastasis. Currently there is no effective drug treatment. While the selective COX-2 inhibitor celecoxib showed anti-neoplastic activity against several malignant tumors, its effects against LGG remain to be elucidated. Ours is the first report that the expression level of COX-2 in brain tissue samples from patients with LGG and in LGG cell lines is higher than in the non-neoplastic region and in normal brain cells. We found that celecoxib attenuated LGG cell proliferation in a dose-dependent manner. It inhibited the generation of prostaglandin E2 and induced apoptosis and cell-cycle arrest. We also show that celecoxib hampered the activation of the Akt/survivin- and the Akt/ID3 pathway in LGGs. These findings suggest that celecoxib may have a promising therapeutic potential and that the early treatment of LGG patients with the drug may be beneficial.

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Development of Highly Selective Fluorescent Probe Enabling Flow-Cytometric Isolation of ALDH3A1-Positive Viable Cells

Yagishita Atsushi, Ueno Tasuku, Esumi Hiroyasu, Saya Hideyuki, Kaneko Kazuhiro, Tsuchihara Katsuya, Urano Yasuteru

Bioconjugate Chemistry 28 ( 2 ) 302 - 306 2017.02

ISSN 1043-1802

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Simvastatin-induced apoptosis in osteosarcoma cells

Kamel Walied A., Sugihara Eiji, Nobusue Hiroyuki, Yamaguchi-Iwai Sayaka, Onishi Nobuyuki, Maki Kenta, Fukuchi Yumi, Matsuo Koichi, Muto Akihiro, Saya Hideyuki, Shimizu Takatsune

Molecular Cancer Therapeutics 16 ( 1 ) 182 - 192 2017.01

ISSN 1535-7163

Osteosarcoma is the most common type of primary bone tumor, novel therapeutic agents for which are urgently needed. To identify such agents, we screened a panel of approved drugs with a mouse model of osteosarcoma. The screen identified simvastatin, which inhibited the proliferation and migration of osteosarcoma cells in vitro. Simvastatin also induced apoptosis in osteosarcoma cells in a manner dependent on inhibition of the mevalonate biosynthetic pathway. It also disrupted the function of the small GTPase RhoA and induced activation of AMP-activated protein kinase (AMPK) and p38 MAPK, with AMPK functioning upstream of p38 MAPK. Inhibitors of AMPK or p38 MAPK attenuated the induction of apoptosis by simvastatin, whereas metformin enhanced this effect of simvastatin by further activation of AMPK. Although treatment with simvastatin alone did not inhibit osteosarcoma tumor growth in vivo, its combination with a fat-free diet induced a significant antitumor effect that was enhanced further by metformin administration. Our findings suggest that simvastatin induces apoptosis in osteosarcoma cells via activation of AMPK and p38 MAPK, and that, in combination with other approaches, it holds therapeutic potential for osteosarcoma.

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Phase I study of salazosulfapyridine in combination with cisplatin and pemetrexed for advanced non-small-cell lung cancer

Otsubo Kohei, Nosaki Kaname, Imamura Chiyo K., Ogata Hiroaki, Fujita Akitaka, Sakata Shinya, Hirai Fumihiko, Toyokawa Gouji, Iwama Eiji, Harada Taishi, Seto Takashi, Takenoyama Mitsuhiro, Ozeki Takeshi, Mushiroda Taisei, Inada Mieko, Kishimoto Junji, Tsuchihashi Kenji, Suina Kentaro, Nagano Osamu, Saya Hideyuki, Nakanishi Yoichi, Okamoto Isamu

Cancer Science 2017

ISSN 1347-9032

Spliced variant isoforms of CD44 (CD44v) are a marker of cancer stem cells in solid tumors. They stabilize the xCT subunit of the transporter system xc(-) and thereby promote synthesis of the antioxidant glutathione. Salazosulfapyridine (SASP) is an inhibitor of xCT and suppresses the proliferation of CD44v-positive cancer cells. Chemotherapy-naïve patients with advanced non-squamous non-small-cell lung cancer were enrolled in a dose-escalation study (standard 3 + 3 design) of SASP in combination with cisplatin and pemetrexed. The primary end-point was the percentage of patients who experience dose-limiting toxicity. Fifteen patients were enrolled in the study. Dose-limiting toxicity was observed in one of six patients at a SASP dose of 1.5 g/day (elevation of aspartate and alanine aminotransferase levels, each of grade 3), two of five patients at 3 g/day (hypotension or pneumonitis, each of grade 3), and two of three patients at 4.5 g/day (anorexia of grade 3). The maximum tolerated dose was thus 3 g/day, and the recommended dose was 1.5 g/day. The overall response rate was 26.7% and median progression-free survival was 11.7 months, much longer than that for cisplatin-pemetrexed alone in previous studies. Exposure to SASP varied markedly among individuals according to ABCG2 and NAT2 genotypes. The serum concentration of free CD44v protein was increased after the first cycle of treatment, possibly reflecting death of cancer stem cells. Salazosulfapyridine was thus given safely in combination with cisplatin-pemetrexed, with the addition of SASP tending to prolong progression-free survival. This trial is registered in the UMIN Clinical Trials Registry as UMIN000017854.

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Distinct metaplastic and inflammatory phenotypes in autoimmune and adenocarcinoma-associated chronic atrophic gastritis

Jeong Sangho, Choi Eunyoung, Petersen Christine P., Roland Joseph T., Federico Alessandro, Ippolito Rossana, D'Armiento Francesco P., Nardone Gerardo, Nagano Osamu, Saya Hideyuki, Romano Marco, Goldenring James R.

United European Gastroenterology Journal 5 ( 1 ) 37 - 44 2017

ISSN 2050-6406

Background: Autoimmune gastritis (AIG) and adenocarcinoma-associated chronic atrophic gastritis (CAG) are both associated with oxyntic atrophy, but AIG patients demonstrate an increased risk of carcinoid tumors rather than the elevated risk of adenocarcinoma observed with CAG. We therefore sought to compare the characteristics of the metaplastic mucosa in AIG and CAG patients. Methods: We examined markers for metaplasia (spasmolytic polypeptide expressing metaplasia (SPEM) and intestinal metaplasia) as well as proliferation (Ki67) and immune cell populations (neutrophils, macrophages, and eosinophils) in gastric sections from 16 female patients with autoimmune thyroiditis and AIG and 17 patients with CAG associated with gastric adenocarcinoma. Results: Both AIG and CAG patients demonstrated prominent SPEM and intestinal metaplasia. However, AIG patients displayed significantly lower numbers of infiltrating macrophages and significantly reduced mucosal cell proliferation as compared to CAG patients. Conclusions: These findings indicate that, while both AIG and CAG patients display prominent oxyntic atrophy and metaplasia, the AIG patients do not show proliferative metaplastic lineages that would predispose to adenocarcinoma.

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RNA Sequencing Analysis Reveals Interactions between Breast Cancer or Melanoma Cells and the Tissue Microenvironment during Brain Metastasis

Sato Ryo, Nakano Teppei, Hosonaga Mari, Sampetrean Oltea, Harigai Ritsuko, Sasaki Takashi, Koya Ikuko, Okano Hideyuki, Kudoh Jun, Saya Hideyuki, Arima Yoshimi

BioMed Research International 2017 2017

ISSN 2314-6133

Metastasis is the main cause of treatment failure and death in cancer patients. Metastasis of tumor cells to the brain occurs frequently in individuals with breast cancer, non-small cell lung cancer, or melanoma. Despite recent advances in our understanding of the causes and in the treatment of primary tumors, the biological and molecular mechanisms underlying the metastasis of cancer cells to the brain have remained unclear. Metastasizing cancer cells interact with their microenvironment in the brain to establish metastases. We have now developed mouse models of brain metastasis based on intracardiac injection of human breast cancer or melanoma cell lines, and we have performed RNA sequencing analysis to identify genes in mouse brain tissue and the human cancer cells whose expression is associated specifically with metastasis. We found that the expressions of the mouse genes Tph2, Sspo, Ptprq, and Pole as well as those of the human genes CXCR4, PLLP, TNFSF4, VCAM1, SLC8A2, and SLC7A11 were upregulated in brain tissue harboring metastases. Further characterization of such genes that contribute to the establishment of brain metastases may provide a basis for the development of new therapeutic strategies and consequent improvement in the prognosis of cancer patients.

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The impact of EpCAM expression on response to chemotherapy and clinical outcomes in patients with epithelial ovarian cancer

Tayama Shingo, Motohara Takeshi, Narantuya Dashdemberel, Li Chenyan, Fujimoto Koichi, Sakaguchi Isao, Tashiro Hironori, Saya Hideyuki, Nagano Osamu, Katabuchi Hidetaka

Oncotarget 8 ( 27 ) 44312 - 44325 2017

ISSN 1949-2553

Epithelial ovarian cancer is a highly lethal malignancy; moreover, overcoming chemoresistance is the major challenging in treating ovarian cancer patients. The cancer stem cell (CSC) hypothesis considers CSCs to be the main culprits in driving tumor initiation, metastasis, and resistance to conventional therapy. Although growing evidence suggest that CSCs are responsible for chemoresistance, the contribution of CSC marker EpCAM to resistance to chemotherapy remains unresolved. Here we have demonstrated that ovarian cancers containing high levels of EpCAM have a significantly much lower probability of achieving overall responsive rates after first-line chemotherapy. In addition, multivariate analysis revealed that EpCAM expression is an independent risk factor for chemoresistance, indicating that EpCAM expression is a predictive biomarker of chemotherapeutic response. Consistent with these clinical observations, in vitro assays, we found that the subpopulation of EpCAMpositive ovarian cancer cells shows a significantly higher viability compared with EpCAM-negative cells in response to cisplatin treatment by preventing chemotherapyinduced apoptosis, which is regulated by EpCAM-Bcl-2 axis. Furthermore, in an in vivo mouse model, platinum agents preferentially eliminated EpCAM-negative cells in comparison with EpCAM-positive cells, suggesting that the remaining subpopulation of EpCAM-positive cells contributes to tumor recurrence after chemotherapy. Finally, we also found that an increased expression of EpCAM is associated with poor prognosis in ovarian cancer patients. Our findings highlight the clinical significance of EpCAM in the resistance to chemotherapy and provide a rationale for EpCAM-targeted therapy to improve chemoresistance. Targeting EpCAM should be a promising approach to effectively extirpate the CSCs as the putative root of ovarian cancer.

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ALK ^R1275Q perturbs extracellular matrix, enhances cell invasion and leads to the development of neuroblastoma in cooperation with MYCN

Ueda T., Nakata Y., Yamasaki N., Oda H., Sentani K., Kanai A., Onishi N., Ikeda K., Sera Y., Honda Z. I., Tanaka K., Sata M., Ogawa S., Yasui W., Saya H., Takita J., Honda H.

Oncogene 35 ( 34 ) 4447 - 4458 2016.08

ISSN 0950-9232

Overexpression of MYCN is a hallmark of neuroblastoma (NB). ALK R1275Q, an activating mutation of ALK (anaplastic lymphoma kinase), has been found in sporadic and familial NB patients. In this report, we demonstrated that ALK R1275Q knock-in, MYCN transgenic compound mice developed NB with complete penetrance. Transcriptome analysis revealed that ALK R1275Q globally downregulated the expression of extracellular matrix (ECM)- and basement membrane (BM)-associated genes in both primary neuronal cells and NB tumors. Accordingly, ALK R1275Q /MYCN tumors exhibited reduced expression of ECM/BM-related proteins as compared with MYCN tumors. In addition, on MYCN transduction, ALK R1275Q -expressing neuronal cells exhibited increased migratory and invasive activities. Consistently, enhanced invasion and metastasis were demonstrated in ALK R1275Q /MYCN mice. These results collectively indicate that ALK R1275Q confers a malignant potential on neuronal cells that overexpress MYCN by impairing normal ECM/BM integrity and enhancing tumor growth and dissemination. Moreover, we found that crizotinib, an ALK inhibitor, almost completely inhibited the growth of ALK R1275Q /MYCN tumors in an allograft model. Our findings provided insights into the cooperative mechanism of the mutated ALK and overexpressed MYCN in the pathogenesis of NB and demonstrated the effectiveness of crizotinib on ALK R1275Q -positive tumors.

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Addiction to the IGF2-ID1-IGF2 circuit for maintenance of the breast cancer stem-like cells

Tominaga K., Shimamura T., Kimura N., Murayama T., Matsubara D., Kanauchi H., Niida A., Shimizu S., Nishioka K., Tsuji E. I., Yano M., Sugano S., Shimono Y., Ishii H., Saya H., Mori M., Akashi K., Tada K. I., Ogawa T., Tojo A., Miyano S., Gotoh N.

Oncogene 2016.08

ISSN 0950-9232

The transcription factor nuclear factor-κB (NF-κB) has important roles for tumorigenesis, but how it regulates cancer stem cells (CSCs) remains largely unclear. We identified insulin-like growth factor 2 (IGF2) is a key target of NF-κB activated by HER2/HER3 signaling to form tumor spheres in breast cancer cells. The IGF2 receptor, IGF1 R, was expressed at high levels in CSC-enriched populations in primary breast cancer cells. Moreover, IGF2-PI3K (IGF2-phosphatidyl inositol 3 kinase) signaling induced expression of a stemness transcription factor, inhibitor of DNA-binding 1 (ID1), and IGF2 itself. ID1 knockdown greatly reduced IGF2 expression, and tumor sphere formation. Finally, treatment with anti-IGF1/2 antibodies blocked tumorigenesis derived from the IGF1R^high CSC-enriched population in a patient-derived xenograft model. Thus, NF-κB may trigger IGF2-ID1-IGF2-positive feedback circuits that allow cancer stem-like cells to appear. Then, they may become addicted to the circuits. As the circuits are the Achilles’ heels of CSCs, it will be critical to break them for eradication of CSCs.Oncogene advance online publication, 22 August 2016; doi:10.1038/onc.2016.293.

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Isolation of all CD44 transcripts in human epidermis and regulation of their expression by various agents

Teye Kwesi, Numata Sanae, Ishii Norito, Krol Rafal P., Tsuchisaka Atsunari, Hamada Takahiro, Koga Hiroshi, Karashima Tadashi, Ohata Chika, Tsuruta Daisuke, Saya Hideyuki, Haftek Marek, Hashimoto Takashi

PLoS One 11 ( 8 ) 2016.08

ISSN 1932-6203

CD44, a cell surface proteoglycan, is involved in many biological events. CD44 transcripts undergo complex alternative splicing, resulting in many functionally distinct isoforms. To date, however, the nature of these isoforms in human epidermis has not been adequately determined. In this study, we isolated all CD44 transcripts from normal human epidermis, and studied how their expressions are regulated. By RT-PCR, we found that a number of different CD44 transcripts were expressed in human epidermis, and we obtained all these transcripts from DNA bands in agarose and acrylamide gels by cloning. Detailed sequence analysis revealed 18 CD44 transcripts, 3 of which were novel. Next, we examined effects of 10 different agents on the expression of CD44 transcripts in cultured human keratinocytes, and found that several agents, particularly epidermal growth factor, hydrogen peroxide, phorbol 12-myristate 13-acetate, retinoic acid, calcium and fetal calf serum differently regulated their expressions in various patterns. Furthermore, normal and malignant keratinocytes were found to produce different CD44 transcripts upon serum stimulation and subsequent starvation, suggesting that specific CD44 isoforms are involved in tumorigenesis via different CD44-mediated biological pathways.

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Concise Review: Stem cells and epithelial-mesenchymal transition (EMT) in cancer: biological implications and therapeutic targets.

Sato Ryo, Semba Takashi, Saya Hideyuki, Arima Yoshimi

Stem Cells 34 ( 8 ) 1997 - 2007 2016.08

ISSN 1066-5099

Cancer stem cells (CSCs) constitute a small subpopulation of cancer cells with stem-like properties that are able to self-renew, generate differentiated daughter cells, and give rise to heterogeneous tumor tissue. Tumor heterogeneity is a hallmark of cancer and underlies resistance to anticancer therapies and disease progression. The epithelial-mesenchymal transition (EMT) is a reversible phenomenon that is mediated by EMT-inducing transcription factors (EMT-TFs) and plays an important role in normal organ development, wound healing, and the invasiveness of cancer cells. Recent evidence showing that overexpression of several EMT-TFs is associated with stemness in cancer cells has suggested the existence of a link between EMT and CSCs. In this review, we focus on the roles of CSCs and EMT signaling in driving tumor heterogeneity. A better understanding of the dynamics of both CSCs and EMT-TFs in the generation of tumor heterogeneity may provide a basis for the development of new treatment options for cancer patients. Stem Cells 2016;34:1997–2007.

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CD44 variant-dependent redox status regulation in liver fluke-associated cholangiocarcinoma

Thanee Malinee, Loilome Watcharin, Techasen Anchalee, Sugihara Eiji, Okazaki Shogo, Abe Shinya, Ueda Shiho, Masuko Takashi, Namwat Nisana, Khuntikeo Narong, Titapun Attapol, Pairojkul Chawalit, Saya Hideyuki, Yongvanit Puangrat

Cancer Science 107 ( 7 ) 991 - 1000 2016.07

ISSN 1347-9032

Expression of CD44, especially the variant isoforms (CD44v) of this major cancer stem cell marker, contributes to reactive oxygen species (ROS) defense through stabilizing xCT (a cystine–glutamate transporter) and promoting glutathione synthesis. This enhances cancer development and increases chemotherapy resistance. We investigate the role of CD44v in the regulation of the ROS defense system in cholangiocarcinoma (CCA). Immunohistochemical staining of CD44v and p38^MAPK (a major ROS target) expression in Opisthorchis viverrini-induced hamster CCA tissues (at 60, 90, 120, and 180 days) reveals a decreased phospho-p38^MAPK signal, whereas the CD44v signal was increased during bile duct transformation. Patients with CCA showed CD44v overexpression and negative-phospho-p38^MAPK patients a significantly shorter survival rate than the low CD44v signal and positive-phospho-p38^MAPK patients (P = 0.030). Knockdown of CD44 showed that xCT and glutathione levels were decreased, leading to a high level of ROS. We examined xCT-targeted CD44v cancer stem cell therapy using sulfasalazine. Glutathione decreased and ROS increased after the treatment, leading to inhibition of cell proliferation and induction of cell death. Thus, the accumulation of CD44v leads to the suppression of p38^MAPK in transforming bile duct cells. The redox status regulation of CCA cells depends on the expression of CD44v to contribute the xCT function and is a link to the poor prognosis of patients. Thus, an xCT inhibitor could inhibit cell growth and activate cell death. This suggests that an xCT-targeting drug may improve CCA therapy by sensitization to the available drug (e.g. gemcitabine) by blocking the mechanism of the cell's ROS defensive system.

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LATS-YAP/TAZ controls lineage specification by regulating TGFβ signaling and Hnf4α expression during liver development

Lee Da Hye, Park Jae Oh, Kim Tae Shin, Kim Sang Kyum, Kim Tack Hoon, Kim Min Chul, Park Gun Soo, Kim Jeong Hwan, Kuninaka Shinji, Olson Eric N., Saya Hideyuki, Kim Seon Young, Lee Ho, Lim Dae Sik

Nature Communications 7 2016.06

ISSN 2041-1723

The Hippo pathway regulates the self-renewal and differentiation of various adult stem cells, but its role in cell fate determination and differentiation during liver development remains unclear. Here we report that the Hippo pathway controls liver cell lineage specification and proliferation separately from Notch signalling, using mice and primary hepatoblasts with liver-specific knockout of Lats1 and Lats2 kinase, the direct upstream regulators of YAP and TAZ. During and after liver development, the activation of YAP/TAZ induced by loss of Lats1/2 forces hepatoblasts or hepatocytes to commit to the biliary epithelial cell (BEC) lineage. It increases BEC and fibroblast proliferation by up-regulating TGFβ signalling, but suppresses hepatoblast to hepatocyte differentiation by repressing Hnf4α expression. Notably, oncogenic YAP/TAZ activation in hepatocytes induces massive p53-dependent cell senescence/death. Together, our results reveal that YAP/TAZ activity levels govern liver cell differentiation and proliferation in a context-dependent manner.

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CD44 splice variant v8-10 as a marker of serous ovarian cancer prognosis

Sosulski Amanda, Horn Heiko, Zhang Lihua, Coletti Caroline, Vathipadiekal Vinod, Castro Cesar M., Birrer Michael J., Nagano Osamu, Saya Hideyuki, Lage Kasper, Donahoe Patricia K., Pépin David

PLoS One 11 ( 6 ) 2016.06

ISSN 1932-6203

CD44 is a transmembrane hyaluronic acid receptor gene that encodes over 100 different tissue-specific protein isoforms. The most ubiquitous, CD44 standard, has been used as a cancer stem cell marker in ovarian and other cancers. Expression of the epithelial CD44 variant containing exons v8-10 (CD44v8-10) has been associated with more chemoresistant and metastatic tumors in gastrointestinal and breast cancers, but its role in ovarian cancer is unknown; we therefore investigated its use as a prognostic marker in this disease. The gene expression profiles of 254 tumor samples from The Cancer Genome Atlas RNAseqV2 were analyzed for the presence of CD44 isoforms. A trend for longer survival was observed in patients with high expression of CD44 isoforms that include exons v8-10. Immunohistochemical (IHC) analysis of tumors for presence of CD44v8-10 was performed on an independent cohort of 210 patients with high-grade serous ovarian cancer using a tumor tissue microarray. Patient stratification based on software analysis of staining revealed a statistically significant increase in survival in patients with the highest levels of transmembrane protein expression (top 10 or 20%) compared to those with the lowest expression (bottom 10 and 20%) (p = 0.0181, p = 0.0262 respectively). Expression of CD44v8-10 in primary ovarian cancer cell lines was correlated with a predominantly epithelial phenotype characterized by high expression of epithelial markers and low expression of mesenchymal markers by qPCR, Western blot, and IHC. Conversely, detection of proteolytically cleaved and soluble extracellular domain of CD44v8-10 in patient ascites samples was correlated with significantly worse prognosis (p

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Eradication of CD44-variant positive population in head and neck tumors through controlled intracellular navigation of cisplatin-loaded nanomedicines

Wang Ming, Miura Yutaka, Tsuchihashi Kenji, Miyano Kazuki, Nagano Osamu, Yoshikawa Momoko, Tanabe Ami, Makino Jun, Mochida Yuki, Nishiyama Nobuhiro, Saya Hideyuki, Cabral Horacio, Kataoka Kazunori

Journal of Controlled Release 230 26 - 33 2016.05

ISSN 0168-3659

Eventual relapse of tumor growth is commonly observed in head and neck cancer patients, following treatment with platinum-based chemotherapies. This occurrence is believed to be related to the failure to eradicate drug resistant, cancer stem cell (CSC) niches, thereby enriching their population in tumors after treatment. In this study, we show that in contrast to free cisplatin (CDDP), the polymer micelle-based nanomedicine incorporating cisplatin (CDDP/m), can eradicate both the undifferentiated cell and the differentiated cancer cell populations within a head and neck tumor model. Immunohistochemistry of treated tumors showed that opposing to CDDP treatment, CDDP/m could reduce tumor growth without concentrating the CSC-like population. We further showed that CDDP/m, but not CDDP, can localize into hypoxic regions, possibly CSC-rich areas, in the tumors, and can overcome their detoxification mechanism based-on high cellular expression of glutathione to successfully deliver Pt to nuclear DNA. Our data suggests CDDP/m to be a replacement for current platinum therapies, for its ability to eradicate both bulk and CSC-like populations, and in turn to prevent recurrence of tumor growth.

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The EGF receptor promotes the malignant potential of glioma by regulating amino acid transport system xc(-)

Tsuchihashi Kenji, Okazaki Shogo, Ohmura Mitsuyo, Ishikawa Miyuki, Sampetrean Oltea, Onishi Nobuyuki, Wakimoto Hiroaki, Yoshikawa Momoko, Seishima Ryo, Iwasaki Yoshimi, Morikawa Takayuki, Abe Shinya, Takao Ayumi, Shimizu Misato, Masuko Takashi, Nagane Motoo, Furnari Frank B., Akiyama Tetsu, Suematsu Makoto, Baba Eishi, Akashi Koichi, Saya Hideyuki, Nagano Osamu

Cancer Research 76 ( 10 ) 2954 - 2963 2016.05

ISSN 0008-5472

Extracellular free amino acids contribute to the interaction between a tumor and its microenvironment through effects on cellular metabolism and malignant behavior. System xc(-) is composed of xCT and CD98hc subunits and functions as a plasma membrane antiporter for the uptake of extracellular cystine in exchange for intracellular glutamate. Here, we show that the EGFR interacts with xCT and thereby promotes its cell surface expression and function inhumanglioma cells. EGFR-expressing glioma cells manifested both enhanced antioxidant capacity as a result of increased cystine uptake, as well as increased glutamate, which promotes matrix invasion. Imaging mass spectrometry also revealed that brain tumors formed in mice by human glioma cells stably overexpressing EGFR contained higher levels of reduced glutathione compared with those formed by parental cells. Targeted inhibition of xCT suppressed the EGFR-dependent enhancement of antioxidant capacity in glioma cells, as well as tumor growth and invasiveness. Our findings establish a new functional role for EGFR in promoting the malignant potential of glioma cells through interaction with xCT at the cell surface.

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Tumour resistance in induced pluripotent stem cells derived from naked mole-rats

Miyawaki Shingo, Kawamura Yoshimi, Oiwa Yuki, Shimizu Atsushi, Hachiya Tsuyoshi, Bono Hidemasa, Koya Ikuko, Okada Yohei, Kimura Tokuhiro, Tsuchiya Yoshihiro, Suzuki Sadafumi, Onishi Nobuyuki, Kuzumaki Naoko, Matsuzaki Yumi, Narita Minoru, Ikeda Eiji, Okanoya Kazuo, Seino Ken Ichiro, Saya Hideyuki, Okano Hideyuki, Miura Kyoko

Nature Communications 7 2016.05

The naked mole-rat (NMR, Heterocephalus glaber), which is the longest-lived rodent species, exhibits extraordinary resistance to cancer. Here we report that NMR somatic cells exhibit a unique tumour-suppressor response to reprogramming induction. In this study, we generate NMR-induced pluripotent stem cells (NMR-iPSCs) and find that NMR-iPSCs do not exhibit teratoma-forming tumorigenicity due to the species-specific activation of tumour-suppressor alternative reading frame (ARF) and a disruption mutation of the oncogene ES cell-expressed Ras (ERAS). The forced expression of Arf in mouse iPSCs markedly reduces tumorigenicity. Furthermore, we identify an NMR-specific tumour-suppression phenotype - ARF suppression-induced senescence (ASIS) - that may protect iPSCs and somatic cells from ARF suppression and, as a consequence, tumorigenicity. Thus, NMR-specific ARF regulation and the disruption of ERAS regulate tumour resistance in NMR-iPSCs. Our findings obtained from studies of NMR-iPSCs provide new insight into the mechanisms of tumorigenicity in iPSCs and cancer resistance in the NMR.

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MIF maintains the tumorigenic capacity of brain tumor-initiating cells by directly inhibiting p53

Fukaya Raita, Ohta Shigeki, Yaguchi Tomonori, Matsuzaki Yumi, Sugihara Eiji, Okano Hideyuki, Saya Hideyuki, Kawakami Yutaka, Kawase Takeshi, Yoshida Kazunari, Toda Masahiro

Cancer Research 76 ( 9 ) 2813 - 2823 2016.05

ISSN 0008-5472

Tumor-initiating cells thought to drive brain cancer are embedded in a complex heterogeneous histology. In this study, we isolated primary cells from 21 human brain tumor specimens to establish cell lines with high tumorigenic potential and to identify the molecules enabling this capability. The morphology, sphereforming ability upon expansion, and differentiation potential of all cell lines were indistinguishable in vitro. However, testing for tumorigenicity revealed two distinct cell types, brain tumor-initiating cells (BTIC) and non-BTIC. We found that macrophage migration inhibitory factor (MIF) was highly expressed in BTIC compared with non-BTIC. MIF bound directly to both wild-type and mutant p53 but regulated p53-dependent cell growth by different mechanisms, depending on glioma cell line and p53 status. MIF physically interacted with wild-type p53 in the nucleus and inhibited its transcription-dependent functions. In contrast, MIF bound to mutant p53 in the cytoplasm and abrogated transcription-independent induction of apoptosis. Furthermore, MIF knockdown inhibited BTIC-induced tumor formation in a mouse xenograft model, leading to increased overall survival. Collectively, our findings suggest that MIF regulates BTIC function through direct, intracellular inhibition of p53, shedding light on the molecular mechanisms underlying the tumorigenicity of certain malignant brain cells.

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Potential of sulfasalazine as a therapeutic sensitizer for CD44 splice variant 9-positive urogenital cancer

Takayama Tatsuya, Kubo Taro, Morikawa Ai, Morita Tatsuo, Nagano Osamu, Saya Hideyuki

Medical Oncology 33 ( 5 ) 2016.05

ISSN 1357-0560

Cancer stem-like cells (CSCs) with high expression of CD44 splice variant (CD44v) have an enhanced capacity for intracellular reduced glutathione synthesis and defense against reactive oxygen species, resulting in resistance to various therapeutic stresses. Sulfasalazine (SSZ), a drug used in the treatment of rheumatoid arthritis (RA), inhibits glutamate–cystine transport, and suppressed CD44v-dependent tumor growth and increased sensitivity to cytotoxic drugs in an in vivo study. Here, we present two cases of CD44v9-positive urogenital cancer with concomitant treatment with SSZ for RA. Patient 1 was a 62-year-old man who had received SSZ for RA beginning 2 months before the diagnosis of urinary bladder cancer. Although he had multiple metastases to the bladder, abdominal, left cervical and left axillary lymph nodes, and brain, complete response with multidisciplinary therapy was maintained for more than 2 years. Patient 2 was a 74-year-old man with castration-resistant prostate cancer who was diagnosed with RA during chemotherapy and a gradual increase in prostate-specific antigen (PSA) level. When SSZ was added, his PSA value (ng/mL) decreased from 12.93 to 5.58 in only 2 weeks and then quickly rebounded, whereas levels of neuron-specific enolase, a neuroendocrine differentiator and CSC marker, remained almost unchanged. We therefore speculate that SSZ treatment may represent a new adjuvant treatment option for patients with CD44v9-positive urogenital cancer.

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Generation of heterozygous fibrillin-1 mutant cloned pigs from genome-edited foetal fibroblasts

Umeyama Kazuhiro, Watanabe Kota, Watanabe Masahito, Horiuchi Keisuke, Nakano Kazuaki, Kitashiro Masateru, Matsunari Hitomi, Kimura Tokuhiro, Arima Yoshimi, Sampetrean Oltea, Nagaya Masaki, Saito Masahiro, Saya Hideyuki, Kosaki Kenjiro, Nagashima Hiroshi, Matsumoto Morio

Scientific Reports 6 2016.04

Marfan syndrome (MFS) is an autosomal dominant genetic disease caused by abnormal formation of the extracellular matrix with an incidence of 1 in 3, 000 to 5, 000. Patients with Marfan syndrome experience poor quality of life caused by skeletal disorders such as scoliosis, and they are at high risk of sudden death from cardiovascular impairment. Suitable animal models of MFS are essential for conquering this intractable disease. In particular, studies employing pig models will likely provide valuable information that can be extrapolated to humans because of the physiological and anatomical similarities between the two species. Here we describe the generation of heterozygous fibrillin-1 (FBN1) mutant cloned pigs (+/Glu433AsnfsX98) using genome editing and somatic cell nuclear transfer technologies. The FBN1 mutant pigs exhibited phenotypes resembling those of humans with MFS, such as scoliosis, pectus excavatum, delayed mineralization of the epiphysis and disrupted structure of elastic fibres of the aortic medial tissue. These findings indicate the value of FBN1 mutant pigs as a model for understanding the pathogenesis of MFS and for developing treatments.

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Dose-escalation study for the targeting of CD44v⁺ cancer stem cells by sulfasalazine in patients with advanced gastric cancer (EPOC1205)

Shitara Kohei, Doi Toshihiko, Nagano Osamu, Imamura Chiyo K., Ozeki Takeshi, Ishii Yuya, Tsuchihashi Kenji, Takahashi Shunji, Nakajima Takako E., Hironaka Shuichi, Fukutani Miki, Hasegawa Hiromi, Nomura Shogo, Sato Akihiro, Einaga Yasuaki, Kuwata Takeshi, Saya Hideyuki, Ohtsu Atsushi

Gastric Cancer 1 - 9 2016.04

ISSN 1436-3291

Background: Cancer stem cells (CSCs) have enhanced mechanisms of protection from oxidative stress. A variant form of CD44 (CD44v), a major CSC marker, was shown to interact with xCT, a subunit of cystine–glutamate transporter, which maintains high levels of intracellular reduced glutathione (GSH) which defend the cell against oxidative stress. Sulfasalazine (SSZ) is an inhibitor of xCT and was shown to suppress the survival of CD44v-positive stem-like cancer cells both in vitro and in vivo. To find the dose of SSZ which can safely reduce the population of CD44v-positive cells in tumors, a dose-escalation study in patients with advanced gastric cancer was conducted. Methods: SSZ was given four times daily by oral administration with 2 weeks as one cycle. Tumor biopsies were obtained before and after 14 days of administration of SSZ to evaluate expression of CD44v and the intratumoral level of GSH. Results: Eleven patients were enrolled and received a dosage from 8 to 12 g/day. Safety was confirmed up to a dosage of 12 g/day, which was considered the maximum tolerated dose. Among the eight patients with CD44v-positive cells in their pretreatment biopsy samples, the CD44v-positive cancer cell population appeared to be reduced in the posttreatment biopsy tissues of four patients. Intratumoral GSH levels were also decreased in two patients, suggesting biological effectiveness of SSZ at 8 g/day or greater. Conclusions: This is the first study of SSZ as an xCT inhibitor for targeting CSCs. Reduction of the levels of CD44v-positive cells and GSH was observed in some patients, consistent with the mode of action of SSZ in CSCs.

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Myeloid differentiation factor 88 signaling in bone marrow-derived cells promotes gastric tumorigenesis by generation of inflammatory microenvironment

Maeda Yusuke, Echizen Kanae, Oshima Hiroko, Yu Liang, Sakulsak Natthiya, Hirose Osamu, Yamada Yoichi, Taniguchi Tadatsugu, Jenkins Brendan J., Saya Hideyuki, Oshima Masanobu

Cancer Prevention Research 9 ( 3 ) 253 - 263 2016.03

ISSN 1940-6207

It has been established that COX-2 and downstream signaling by prostaglandin E₂ (PGE₂) play a key role in tumorigenesis through generation of inflammatory microenvironment. Tolllike receptor (TLR) signaling through myeloid differentiation factor 88 (MyD88) also regulates inflammatory responses in tumors. However, the relationship between these distinct pathways in tumorigenesis is not yet fully understood. We herein investigated the role of MyD88 in gastric tumorigenesis using Gan mice, which develop inflammation-associated gastric tumors due to the simultaneous activation of the COX-2/PGE₂ pathway and Wnt signaling. Notably, the disruption of Myd88 in Gan mice resulted in the significant suppression of gastric tumorigenesis with the inhibition of inflammatory responses, even though COX-2/PGE₂ pathway is constitutively activated. Moreover, Myd88 disruption in bone marrow-derived cells (BMDCs) in Gan mice also suppressed inflammation and tumorigenesis, indicating that MyD88 signaling in BMDCs regulates the inflammatory microenvironment. We also found that expression of Tlr2 and its coreceptor Cd14 was induced in tumor epithelial cells in Gan mice, which was suppressed by the disruption of Myd88. It has already been shown that TLR2/CD14 signaling is important for stemness of intestinal epithelial cells. These results indicate that MyD88 in BMDCs, together with COX-2/PGE₂ pathway, plays an essential role in the generation of the inflammatory microenvironment, which may promote tumorigenesis through induction of TLR2/CD14 pathway in tumor epithelial cells. These results suggest that inhibition of TLR/MyD88 signaling together with COX-2/PGE₂ pathway will be an effective preventive strategy for gastric cancer.

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Report on the use of non-clinical studies in the regulatory evaluation of oncology drugs

Hayakawa Yoshihiro, Kawada Manabu, Nishikawa Hiroyoshi, Ochiya Takahiro, Saya Hideyuki, Seimiya Hiroyuki, Yao Ryoji, Hayashi Masahiro, Kai Chieko, Matsuda Akira, Naoe Tomoki, Ohtsu Atsushi, Okazaki Taku, Saji Hideo, Sata Masataka, Sugimura Haruhiko, Sugiyama Yuichi, Toi Masakazu, Irimura Tatsuro

Cancer Science 107 ( 2 ) 189 - 202 2016.02

ISSN 1347-9032

Non-clinical studies are necessary at each stage of the development of oncology drugs. Many experimental cancer models have been developed to investigate carcinogenesis, cancer progression, metastasis, and other aspects in cancer biology and these models turned out to be useful in the efficacy evaluation and the safety prediction of oncology drugs. While the diversity and the degree of engagement in genetic changes in the initiation of cancer cell growth and progression are widely accepted, it has become increasingly clear that the roles of host cells, tissue microenvironment, and the immune system also play important roles in cancer. Therefore, the methods used to develop oncology drugs should continuously be revised based on the advances in our understanding of cancer. In this review, we extensively summarize the effective use of those models, their advantages and disadvantages, ranges to be evaluated and limitations of the models currently used for the development and for the evaluation of oncology drugs. This review summarizes the effective use of animal models, their advantages and disadvantages, ranges to be evaluated and limitations of the models currently used for the development and for the evaluation of oncology drugs.

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Development of an ErbB4 monoclonal antibody that blocks neuregulin-1-induced ErbB4 activation in cancer cells

Okazaki Shogo, Nakatani Fumi, Masuko Kazue, Tsuchihashi Kenji, Ueda Shiho, Masuko Takashi, Saya Hideyuki, Nagano Osamu

Biochemical and Biophysical Research Communications 470 ( 1 ) 239 - 244 2016.01

ISSN 0006-291X

The use of monoclonal antibodies (mAbs) for cancer therapy is one of the most important strategies for current cancer treatment. The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, which regulates cancer cell proliferation, survival, and migration, is a major molecular target for antibody-based therapy. ErbB4/HER4, which contains a ligand-binding extracellular region, is activated by several ligands, including neuregulins (NRGs), heparin-binding EGF-like growth factor, betacellulin and epiregulin. Although there are clinically approved antibodies for ErbB1 and ErbB2, there are no available therapeutic mAbs for ErbB4, and it is not known whether ErbB4 is a useful target for antibody-based cancer therapy. In this study, we developed an anti-ErbB4 mAb (clone P6-1) that suppresses NRG-dependent activation of ErbB4 and examined its effect on breast cancer cell proliferation in the extracellular matrix.

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Variant isoforms of CD44 expression in upper tract urothelial cancer as a predictive marker for recurrence and mortality

Hagiwara Masayuki, Kikuchi Eiji, Kosaka Takeo, Mikami Shuji, Saya Hideyuki, Oya Mototsugu

Urologic Oncology: Seminars and Original Investigations 2016.01

ISSN 1078-1439

Purpose: The variant isoforms of CD44 (CD44v), which has been associated with treatment resistance and tumor recurrence, are contributed to the feature of some of the new cell surface markers for cancer stem cells. The aim of this study is to address the prognostic significant of CD44v expression in upper tract urothelial cancer (UTUC). Materials and methods: We retrospectively analyzed the medical records of 110 patients treated surgically for≥pT2 UTUC. To determine the biological significance of CD44v in UTUC, we examined the immunohistochemical expression of CD44v9 and its association with clinical features and oncological outcomes. Results: During the mean follow-up of 4.8 years, tumor recurrence, including local and distant metastasis, was observed in 57 patients (51.8%), and 42 patients (38.2%) died of the disease. The incidence of ureter cancers was significantly higher in patients expressing CD44v9. In this series, 37 patients in the CD44v9-positive group (45.1%) and 5 (17.9%) in the CD44v9-negative group died of the disease. Kaplan-Meier curves revealed that cancer-specific survival and recurrence-free survival rates were significantly lower in the CD44v9-positive group than in the CD44v9-negative group (P = 0.032 and P = 0.038, respectively). A multivariate analysis identified the expression of CD44v9 as one of the independent risk factors for cancer-specific survival (P = 0.040, hazard ratio = 2.67) in addition to tumor grade G3, and also for recurrence-free survival (P = 0.028, hazard ratio = 2.33), in addition to tumor grade G3 and lymphovascular invasion. Conclusions: The expression of CD44v9 may be a new biomarker of malignant potential in muscle invasive UTUC and provide additional prognostic information in patients with UTUC.

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Therapeutic strategies targeting cancer stem cells

Yoshida Go J., Saya Hideyuki

Cancer Science 107 ( 1 ) 5 - 11 2016.01

ISSN 1347-9032

Cancer stem cells (CSCs) are undifferentiated cancer cells with a high tumorigenic activity, the ability to undergo self-renewal, and a multilineage differentiation potential. Cancer stem cells are responsible for the development of tumor cell heterogeneity, a key feature for resistance to anticancer treatments including conventional chemotherapy, radiation therapy, and molecularly targeted therapy. Furthermore, minimal residual disease, the major cause of cancer recurrence and metastasis, is enriched in CSCs. Cancer stem cells also possess the property of "robustness", which encompasses several characteristics including a slow cell cycle, the ability to detoxify or mediate the efflux of cytotoxic agents, resistance to oxidative stress, and a rapid response to DNA damage, all of which contribute to the development of therapeutic resistance. The identification of mechanisms underlying such characteristics and the development of novel approaches to target them will be required for the therapeutic elimination of CSCs and the complete eradication of tumors. In this review, we focus on two prospective therapeutic approaches that target CSCs with the aim of disrupting their quiescence or redox defense capability.

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Development of mouse models of malignant phyllodes tumors by transplantation of syngeneic mammary gland cells expressing mutant H-Ras

Takamoto Yayoi, Arima Yoshimi, Saya Hideyuki

Genes to Cells 2016

ISSN 1356-9597

Phyllodes tumors (PTs) are rare fibroepithelial tumors of the breast with epithelial and stromal components, and surgical resection is the standard and only available treatment for malignant PTs. To provide a better understanding of these tumors, we developed mouse models that recapitulate the pathological and clinical properties of human malignant PTs. Mouse undifferentiated mammary gland cells were infected with a retrovirus encoding the human oncoprotein H-Ras^G12V, and the infected cells were transplanted orthotopically into the mammary fat pads of syngeneic mice. The transplanted cells showed a high tumorigenic activity, with the resulting tumors manifesting pathological characteristics including stromal overgrowth similar to those of human malignant PTs. The tumors also showed high rates of both local recurrence and lung metastasis. Our models may prove useful for studies of the pathophysiology of malignant PTs as well as facilitate the development of new treatments.

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GANP protein encoded on human chromosome 21/mouse chromosome 10 is associated with resistance to mammary tumor development

Kuwahara Kazuhiko, Yamamoto-Ibusuki Mutsuko, Zhang Zhenhuan, Phimsen Suchada, Gondo Naomi, Yamashita Hiroko, Takeo Toru, Nakagata Naomi, Yamashita Daisuke, Fukushima Yoshimi, Yamamoto Yutaka, Iwata Hiroji, Saya Hideyuki, Kondo Eisaku, Matsuo Keitaro, Takeya Motohiro, Iwase Hirotaka, Sakaguchi Nobuo

Cancer Science 2016

ISSN 1347-9032

Human chromosome 21 is known to be associated with the high risk of hematological malignancy but with resistance to breast cancer in the study of Down syndrome. In human cancers, we previously observed the significant alterations of the protein expression encoded by the ganp/MCM3AP gene on human chromosome 21q22.3. Here, we investigated GANP protein alterations in human breast cancer samples (416 cases) at various stages by immunohistochemical analysis. This cohort study clearly showed that expression of GANP is significantly decreased in human breast cancer cases with poor prognosis as an independent risk factor (relapse-free survival, hazard ratio = 2.37, 95% confidence interval, 1.27-4.42, P = 0.007 [univariate analysis]; hazard ratio = 2.70, 95% confidence interval, 1.42-5.13, P = 0.002 [multivariate analysis]). To investigate whether the altered GANP expression is associated with mammary tumorigenesis, we created mutant mice that were conditionally deficient in the ganp/MCM3AP gene using wap-cre recombinase transgenic mice. Mammary gland tumors occurred at a very high incidence in female mammary gland-specific GANP-deficient mice after severe impairment of mammary gland development during pregnancy. Moreover, tumor development also occurred in female post parous GANP-heterodeficient mice. GANP has a significant role in the suppression of DNA damage caused by estrogen in human breast cancer cell lines. These results indicated that the GANP protein is associated with breast cancer resistance.

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CD44 variant 9 is a potential biomarker of tumor initiating cells predicting survival outcome in hepatitis C virus-positive patients with resected hepatocellular carcinoma

Kakehashi Anna, Ishii Naomi, Sugihara Eiji, Gi Min, Saya Hideyuki, Wanibuchi Hideki

Cancer Science 2016

ISSN 1347-9032

This study investigated whether the expression of CD44 variant 9 (CD44v9) might be a functional marker of tumor-initiating stem-like cells in primary hepatocellular carcinomas (HCCs) of hepatitis C virus (HCV)⁺ patients and provide an indicator of patient survival, as well as associated mechanisms. A total of 90 HCV⁺ HCC patients who underwent surgery from 2006 to 2011 were enrolled and monitored for 2-8 years. Expression of CD44v9 was validated immunohistochemically in all HCCs, followed by comparative proteome, survival, and clinicopathological analyses. CD44 variant 8--10 was further evaluated in diethylnitrosamine-induced HCCs of C57Bl/6J mice. Focally localized CD44v⁺ cells with a membranous staining pattern were detected in human HCV⁺ and mouse HCCs. CD44v9⁺ cells of HCCs were predominantly negative for Ki67 and P-p38, indicating decrease of cell proliferation in the CD44v9⁺ tumor cell population, likely to be related to suppression of intracellular oxidative stress due to activation of Nrf2-mediated signaling, DNA repair, and inhibition of xenobiotic metabolism. CD44v9 IHC evaluation in 90 HCV⁺ HCC cases revealed that positive expression was significantly associated with poor overall and recurrence-free survival, a younger age, poor histological differentiation of HCCs, and high alkaline phosphatase levels compared with patients with negative expression. CD44v9 is concluded to be a potential biomarker of tumor-initiating stem-like cells and a prognostic marker in HCV⁺ HCC patients associated with Nrf2-mediated resistance to oxidative stress.

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Antitumor activity of KW-2450 against triple-negative breast cancer by inhibiting Aurora A and B kinases

Kai Kazuharu, Kondo Kimie, Wang Xiaoping, Xie Xuemei, Pitner Mary K., Reyes Monica E., Torres-Adorno Angie M., Masuda Hiroko, Hortobagyi Gabriel N., Bartholomeusz Chandra, Saya Hideyuki, Tripathy Debu, Sen Subrata, Ueno Naoto T.

Molecular Cancer Therapeutics 14 ( 12 ) 2687 - 2699 2015.12

ISSN 1535-7163

Currently, no targeted drug is available for triple-negative breast cancer (TNBC), an aggressive breast cancer that does not express estrogen receptor, progesterone receptor, or HER2. TNBC has high mitotic activity, and, because Aurora A and B mitotic kinases drive cell division and are overexpressed in tumors with a high mitotic index, we hypothesized that inhibiting Aurora A and B produces a significant antitumor effect in TNBC. We tested this hypothesis by determining the antitumor effects of KW- 2450, a multikinase inhibitor of both Aurora A and B kinases. We observed significant inhibitory activities of KW-2450 on cell viability, apoptosis, colony formation in agar, and mammosphere formation in TNBC cells. The growth of TNBC xenografts was significantly inhibited with KW-2450. In cell-cycle analysis, KW-2450 induced tetraploid accumulation followed by apoptosis or surviving octaploid (8N) cells, depending on dose. These phenotypes resembled those of Aurora B knockdown and complete pharmaceutical inhibition of Aurora A. We demonstrated that 8N cells resulting from KW-2450 treatment depended on the activation of mitogen-activated protein kinase kinase (MEK) for their survival. When treated with the MEK inhibitor selumetinib combined with KW-2450, compared with KW-2450 alone, the 8N cell population was significantly reduced and apoptosis was increased. Indeed, this combination showed synergistic antitumor effect in SUM149 TNBC xenografts. Collectively, Aurora A and B inhibition had a significant antitumor effect against TNBC, and this antitumor effect was maximized by the combination of selumetinib with Aurora A and B inhibition.

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Hair follicle-derived IL-7 and IL-15 mediate skin-resident memory T cell homeostasis and lymphoma

Adachi Takeya, Kobayashi Tetsuro, Sugihara Eiji, Yamada Taketo, Ikuta Koichi, Pittaluga Stefania, Saya Hideyuki, Amagai Masayuki, Nagao Keisuke

Nature Medicine 21 ( 11 ) 1272 - 1279 2015.11

ISSN 1078-8956

The skin harbors a variety of resident leukocyte subsets that must be tightly regulated to maintain immune homeostasis. Hair follicles are unique structures in the skin that contribute to skin dendritic cell homeostasis through chemokine production. We demonstrate that CD4 + and CD8 + skin-resident memory T cells (T RM cells), which are responsible for long-term skin immunity, reside predominantly within the hair follicle epithelium of the unperturbed epidermis. T RM cell tropism for the epidermis and follicles is herein termed epidermotropism. Hair follicle expression of IL-15 was required for CD8 + T RM cells, and IL-7 for CD8 + and CD4 + T RM cells, to exert epidermotropism. A lack of either cytokine in the skin led to impaired hapten-induced contact hypersensitivity responses. In a model of cutaneous T cell lymphoma, epidermotropic CD4 + T RM lymphoma cell localization depended on the presence of hair follicle-derived IL-7. These findings implicate hair follicle-derived cytokines as regulators of malignant and non-malignant T RM cell tissue residence, and they suggest that the cytokines may be targeted therapeutically in inflammatory skin diseases and lymphoma.

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Application of clotrimazole via a novel controlled release device provides potent retinal protection

Nezhad Zhaleh Kashkouli, Nagai Nobuhiro, Yamamoto Kotaro, Kaji Hirokazu, Nishizawa Matsuhiko, Saya Hideyuki, Nakazawa Toru, Abe Toshiaki

Journal of Materials Science: Materials in Medicine 26 ( 9 ) 2015.09

ISSN 0957-4530

Age-related macular degeneration is the leading cause of legal blindness among older individuals. Therefore, the development of new therapeutic agents and optimum drug delivery systems for its treatment are crucial. In this study, we investigate whether clotrimazole (CLT) is capable of protecting retinal cells against oxidative-induced injury and the possible inhibitory effect of a sustained CLT-release device against light-induced retinal damage in rats. In vitro results indicated pretreatment of immortalized retinal pigment epithelium cells (RPE-J cells) with 10–50 µM CLT before exposure to oxygen/glucose deprivation conditions for 48 h decreased the extent of cell death, attenuated the percentage of reactive oxygen species-positive cells, and decreased the levels of cleaved caspase-3. The device consists of a separately fabricated reservoir, a CLT formulation, and a controlled release cover, which are made of poly(ethyleneglycol) dimethacrylate (PEGDM) and tri(ethyleneglycol) dimethacrylate (TEGDM). The release rate of CLT was successfully tuned by changing the ratio of PEGDM/TEGDM in the cover. In vivo results showed that use of a CLT-loaded device lessened the reduction of electroretinographic amplitudes after light exposure. These findings indicate that the application of a polymeric CLT-loaded device may be a promising method for the treatment of some retinal disorders.

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Dynamic epigenetic regulation of glioblastoma tumorigenicity through LSD1 modulation of MYC expression

Kozono David, Li Jie, Nitta Masayuki, Sampetrean Oltea, Gonda David, Kushwaha Deepa S., Merzon Dmitry, Ramakrishnan Valya, Zhu Shan, Zhu Kaya, Matsui Hiroko, Harismendy Olivier, Hua Wei, Mao Ying, Kwon Chang Hyuk, Saya Hideyuki, Nakano Ichiro, Pizzo Donald P., Van Den Berg Scott R., Chen Clark C.

Proceedings of the National Academy of Sciences of the United States of America 112 ( 30 ) E4055 - E4064 2015.07

ISSN 0027-8424

The available evidence suggests that the lethality of glioblastoma is driven by small subpopulations of cells that self-renew and exhibit tumorigenicity. It remains unclear whether tumorigenicity exists as a static property of a few cells or as a dynamically acquired property. We used tumor-sphere and xenograft formation as assays for tumorigenicity and examined subclones isolated from established and primary glioblastoma lines. Our results indicate that glioblastoma tumorigenicity is largely deterministic, yet the property can be acquired spontaneously at low frequencies. Further, these dynamic transitions are governed by epigenetic reprogramming through the lysine-specific demethylase 1 (LSD1). LSD depletion increases trimethylation of histone 3 lysine 4 at the avian myelocytomatosis viral oncogene homolog (MYC) locus, which elevates MYC expression. MYC, in turn, regulates oligodendrocyte lineage transcription factor 2(OLIG2), SRY (sex determining region Y)-box 2(SOX2), and POU class 3 homeobox 2 (POU3F2), a core set of transcription factors required for reprogramming glioblastoma cells into stem-like states. Our model suggests epigenetic regulation of key transcription factors governs transitions between tumorigenic states and provides a framework for glioblastoma therapeutic development.

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Methotrexate inhibits osteoclastogenesis by decreasing RANKL-induced calcium influx into osteoclast progenitors

Kanagawa Hiroya, Masuyama Ritsuko, Morita Mayu, Sato Yuiko, Niki Yasuo, Kobayashi Tami, Katsuyama Eri, Fujie Atsuhiro, Hao Wu, Tando Toshimi, Watanabe Ryuichi, Miyamoto Kana, Morioka Hideo, Matsumoto Morio, Toyama Yoshiaki, Saya Hideyuki, Miyamoto Takeshi

Journal of Bone and Mineral Metabolism 2015.07

ISSN 0914-8779

The increasing number of osteoporosis patients is a pressing issue worldwide. Osteoporosis frequently causes fragility fractures, limiting activities of daily life and increasing mortality. Many osteoporosis patients take numerous medicines due to other health issues; thus, it would be preferable if a single medicine could ameliorate osteoporosis and other conditions. Here, we screened 96 randomly selected drugs targeting various diseases for their ability to inhibit differentiation of osteoclasts, which play a pivotal role in development of osteoporosis, and identified methotrexate (MTX), as a potential inhibitor. MTX is currently used to treat sarcomas or leukemic malignancies or auto-inflammatory diseases such as rheumatoid arthritis (RA) through its anti-proliferative and immunosuppressive activities; however, a direct effect on osteoclast differentiation has not been shown. Here, we report that osteoclast formation and expression of osteoclastic genes such as NFATc1 and DC-STAMP, which are induced by the cytokine RANKL, are significantly inhibited by MTX. We found that RANKL-dependent calcium (Ca) influx into osteoclast progenitors was significantly inhibited by MTX. RA patients often develop osteoporosis, and osteoclasts are reportedly required for joint destruction; thus, MTX treatment could have a beneficial effect on RA patients exhibiting high osteoclast activity by preventing both osteoporosis and joint destruction.

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Synergistic antiproliferative effect of imatinib and adriamycin in platelet-derived growth factor receptor-expressing osteosarcoma cells

Yamaguchi Sayaka I., Ueki Arisa, Sugihara Eiji, Onishi Nobuyuki, Yaguchi Tomonori, Kawakami Yutaka, Horiuchi Keisuke, Morioka Hideo, Matsumoto Morio, Nakamura Masaya, Muto Akihiro, Toyama Yoshiaki, Saya Hideyuki, Shimizu Takatsune

Cancer Science 106 ( 7 ) 875 - 882 2015.07

ISSN 1347-9032

Osteosarcoma (OS) is the most frequent primary solid malignant tumor of bone. Its prognosis remains poor in the substantial proportion of patients who do not respond to chemotherapy and novel therapeutic options are therefore needed. We previously established a mouse model that mimics the aggressive behavior of human OS. Enzyme-linked immunosorbent assay-based screening of such mouse tumor lysates identified platelet-derived growth factor-BB (PDGF-BB) as an abundant soluble factor, the gene for which was expressed dominantly in surrounding non-malignant cells of the tumor, whereas that for the cognate receptor (PDGF receptor β) was highly expressed in OS cells. Platelet-derived growth factor-BB induced activation of both MEK-ERK and phosphatidylinositol 3-kinase-protein kinase B signaling pathways and promoted survival in OS cells deprived of serum, and these effects were blocked by the PDGF receptor inhibitor imatinib. However, these actions of PDGF-BB and imatinib were mostly masked in the presence of serum. Whereas imatinib alone did not manifest an antitumor effect in mice harboring OS tumors, combined treatment with imatinib and adriamycin exerted a synergistic antiproliferative effect on OS cells in vivo. These results suggest that treatment of OS with imatinib is effective only when cell survival is dependent on PDGF signaling or when imatinib is combined with another therapeutic intervention that renders the tumor cells susceptible to imatinib action, such as by inducing cellular stress.

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Periostin is a key niche component for wound metastasis of melanoma

Fukuda Keitaro, Sugihara Eiji, Ohta Shoichiro, Izuhara Kenji, Funakoshi Takeru, Amagai Masayuki, Saya Hideyuki

PLoS One 10 ( 6 ) 2015.06

Tissue injury promotes metastasis of several human cancers, although factors associated with wound healing that attract circulating tumor cells have remained unknown. Here, we examined the primary and metastatic lesions that appeared 1 month after trauma in a patient with acral lentiginous melanoma. The levels of mRNA for periostin (POSTN), type 1 collagen, and fibronectin were significantly increased in the metastatic lesion relative to the primary lesion. The increase of these extracellular matrix proteins at the wound site was reproduced in a mouse model of wound healing, with the upregulation of Postn mRNA persisting the longest. POSTN was expressed in the region surrounding melanoma cell nests in metastatic lesions of both wounded mice and the patient. POSTN attenuated the cell adhesion and promoted the migration of melanoma cells without affecting their proliferation in vitro. In the mouse model, the wound site as well as subcutaneously injected osteoblasts that secrete large amounts of POSTN invited the metastasis of remotely-transplanted melanoma cells on the sites. Osteoblasts with suppression of POSTN by shRNA showed a greatly reduced ability to promote such metastasis. Our results suggest that POSTN is a key factor in promoting melanoma cell metastasis to wound sites by providing a premetastatic niche.

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Arsenic trioxide sensitizes glioblastoma to a Myc inhibitor

Yoshimura Yayoi, Shiino Akihiko, Muraki Kazue, Fukami Tadateru, Yamada Shigeki, Satow Takeshi, Fukuda Miyuki, Saiki Masaaki, Hojo Masato, Miyamoto Susumu, Onishi Nobuyuki, Saya Hideyuki, Inubushi Toshiro, Nozaki Kazuhiko, Tanigaki Kenji

PLoS One 10 ( 6 ) 2015.06

Glioblastoma multiforme (GBM) is associated with high mortality due to infiltrative growth and recurrence. Median survival of the patients is less than 15 months, increasing requirements for new therapies. We found that both arsenic trioxide and 10058F4, an inhibitor of Myc, induced differentiation of cancer stem-like cells (CSC) of GBM and that arsenic trioxide drastically enhanced the anti-proliferative effect of 10058F4 but not apoptotic effects. EGFR-driven genetically engineered GBM mouse model showed that this cooperative effect is higher in EGFRvIII-expressing INK4a/Arf-/- neural stem cells (NSCs) than in control wild type NSCs. In addition, treatment of GBM CSC xenografts with arsenic trioxide and 10058F4 resulted in significant decrease in tumor growth and increased differentiation with concomitant decrease of proneural and mesenchymal GBM CSCs in vivo. Our study was the first to evaluate arsenic trioxide and 10058F4 interaction in GBM CSC differentiation and to assess new opportunities for arsenic trioxide and 10058F4 combination as a promising approach for future differentiation therapy of GBM.

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Ink4a/arf-dependent loss of parietal cells induced by oxidative stress promotes CD44-dependent gastric tumorigenesis

Seishima Ryo, Wada Takeyuki, Tsuchihashi Kenji, Okazaki Shogo, Yoshikawa Momoko, Oshima Hiroko, Oshima Masanobu, Sato Toshiro, Hasegawa Hirotoshi, Kitagawa Yuko, Goldenring James R., Saya Hideyuki, Nagano Osamu

Cancer Prevention Research 8 ( 6 ) 492 - 501 2015.06

ISSN 1940-6207

Loss of parietal cells initiates the development of spasmolytic polypeptide-expressing metaplasia (SPEM), a precancerous lesion in stomach. CD44 variant (CD44v) that enhances the ability to defend against reactive oxygen species (ROS) in epithelial cells is expressed de novo in SPEM of K19-Wnt1/C2mE mice, a transgenic model of gastric tumorigenesis, and is required for the efficient development of SPEM and gastric tumor in these animals. The role of ROS and its downstream signaling in CD44-dependent gastric tumorigenesis has remained unknown, however. With the use of the K19-Wnt1/C2mE mouse, we now show that parietal cells in the inflamed stomach are highly sensitive to oxidative stress and manifest activation of p38MAPK signaling by ROS. Oral treatment with the antioxidant ascorbic acid or genetic ablation of the Ink4a/Arf locus, a major downstream target of ROS-p38MAPK signaling, inhibited parietal cell loss and the subsequent gastric tumorigenesis. Our results indicate that signaling activated by oxidative stress in parietal cells plays a key role in CD44-dependent gastric tumorigenesis.

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Impacts of CD44 knockdown in cancer cells on tumor and host metabolic systems revealed by quantitative imaging mass spectrometry

Ohmura Mitsuyo, Hishiki Takako, Yamamoto Takehiro, Nakanishi Tsuyoshi, Kubo Akiko, Tsuchihashi Kenji, Tamada Mayumi, Toue Sakino, Kabe Yasuaki, Saya Hideyuki, Suematsu Makoto

Nitric Oxide - Biology and Chemistry 46 102 - 113 2015.04

ISSN 1089-8603

CD44 expressed in cancer cells was shown to stabilize cystine transporter (xCT) that uptakes cystine and excretes glutamate to supply cysteine as a substrate for reduced glutathione (GSH) for survival. While targeting CD44 serves as a potentially therapeutic stratagem to attack cancer growth and chemoresistance, the impact of CD44 targeting in cancer cells on metabolic systems of tumors and host tissues in vivo remains to be fully determined. This study aimed to reveal effects of CD44 silencing on alterations in energy metabolism and sulfur-containing metabolites in vitro and in vivo using capillary electrophoresis-mass spectrometry and quantitative imaging mass spectrometry (Q-IMS), respectively. In an experimental model of xenograft transplantation of human colon cancer HCT116 cells in superimmunodeficient NOG mice, snap-frozen liver tissues containing metastatic tumors were examined by Q-IMS. As reported previously, short hairpin CD44 RNA interference (shCD44) in cancer cells caused significant regression of tumor growth in the host liver. Under these circumstances, the CD44 knockdown suppressed polyamines, GSH and energy charges not only in metastatic tumors but also in the host liver. In culture, HCT116 cells treated with shCD44 decreased total amounts of methionine-pool metabolites including spermidine and spermine, and reactive cysteine persulfides, suggesting roles of these metabolites for cancer growth. Collectively, these results suggest that CD44 expressed in cancer accounts for a key regulator of metabolic interplay between tumor and the host tissue.

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Expression of TNF-α and CD44 is implicated in poor prognosis, cancer cell invasion, metastasis and resistance to the sunitinib treatment in clear cell renal cell carcinomas

Mikami Shuji, Mizuno Ryuichi, Kosaka Takeo, Saya Hideyuki, Oya Mototsugu, Okada Yasunori

International Journal of Cancer 136 ( 7 ) 1504 - 1514 2015.04

ISSN 0020-7136

Tumor necrosis factor-α (TNF-α) is involved in epithelial-mesenchymal transition (EMT) and expression of CD44, a cancer stem cell marker, in several cancers. This study was performed to clarify the significance of TNF-α and CD44 in clear cell renal cell carcinomas (ccRCCs). Expression of TNF-α and CD44 was examined by immunohistochemistry in 120 ccRCCs. Involvement of TNF-α in EMT and induction of CD44 was analyzed by monitoring expression of EMT-related genes and CD44, and invasion in cultured ccRCC cell lines. TNF-α and CD44 were immunolocalized mainly to carcinoma cells of high-grade ccRCCs with positive correlations with primary tumor stage. A positive correlation was also obtained between TNF-α and CD44 expression, and co-upregulation of TNF-α and CD44 was associated with primary tumor stage, distant metastasis, and poor prognosis. TNF-αenhanced migration and invasion of ccRCC cells together with down-regulation of E-cadherin expression and up-regulation of matrix metalloproteinase 9 and CD44 expression. TNF-α also up-regulated the expression of TNF-α itself in ccRCC cells. Among the 25 ccRCC patients treated with sunitinib for metastatic disease, high CD44 expression was associated with poor treatment outcome. Importantly, residual carcinoma cells in the sunitinib-treated metastatic ccRCCs were strongly positive for CD44, and the CD44 expression was significantly higher in the tumors from the sunitinib-treated patients than in those from untreated ones. Our data show that TNF-α plays an important role in progression of ccRCCs by inducing EMT and CD44 expression, and suggest that CD44 induced by TNF-α may be involved in the resistance to the sunitinib treatment.

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LATS1 and LATS2 phosphorylate CDC26 to modulate assembly of the tetratricopeptide repeat subcomplex of APC/C

Masuda Kenta, Chiyoda Tatsuyuki, Sugiyama Naoyuki, Segura-Cabrera Aldo, Kabe Yasuaki, Ueki Arisa, Banno Koji, Suematsu Makoto, Aoki Daisuke, Ishihama Yasushi, Saya Hideyuki, Kuninaka Shinji

PLoS One 10 ( 2 ) 2015.02

In budding yeast, the Mitotic Exit Network (MEN) regulates anaphase promoting complex/cyclosome (APC/C) via the Dbf2-Cdc14 signaling cascade. Dbf2 kinase phosphorylates and activates Cdc14 phosphatase, which removes the inhibitory phosphorylation of the APC/C cofactor Cdh1. Although each component of the MEN was highly conserved during evolution, there is presently no evidence supporting direct phosphorylation of CDC14 by large tumor suppressor kinase 1 (LATS1), the human counterpart of Dbf2; hence, it is unclear how LATS1 regulates APC/C. Here, we demonstrate that LATS1 phosphorylates the Thr7 (T7) residue of the APC/C component CDC26 directly. Nocodazole-induced phosphorylation of T7 was reduced by knockdown of LATS1 and LATS2 in HeLa cells, indicating that both of these kinases contribute to the phosphorylation of CDC26 in vivo. The T7 residue of CDC26 is critical for its interaction with APC6, a tetratricopeptide repeat-containing subunit of APC/C, and mutation of this residue to Asp (T7D) reduced the interaction of CDC26 with APC6. Replacement of endogenous CDC26 in HeLa cells with exogenous phosphor-mimic T7D-mutated CDC26 increased the elution size of APC/C subunits in a gel filtration assay, implying a change in the APC/C assembly upon phosphorylation of CDC26. Furthermore, T7D-mutated CDC26 promoted the ubiquitination of polo-like kinase 1, a well-known substrate of APC/C. Overall, these results suggest that LATS1/2 are novel kinases involved in APC/C phosphorylation and indicate a direct regulatory link between LATS1/2 and APC/C.

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Sulfasalazine, a therapeutic agent for ulcerative colitis, inhibits the growth of CD44v9⁺ cancer stem cells in ulcerative colitis-related cancer

Seishima Ryo, Okabayashi Koji, Nagano Osamu, Hasegawa Hirotoshi, Tsuruta Masashi, Shimoda Masayuki, Kameyama Kaori, Saya Hideyuki, Kitagawa Yuko

Clinics and Research in Hepatology and Gastroenterology 2015

ISSN 2210-7401

Background and objective: Sulfasalazine reduces the risk of ulcerative colitis (UC)-related cancer through its anti-inflammatory effect and induction of oxidative stress in cancer cells by inhibiting the glutamate-cystine transporter, which is closely associated with the cancer stem cell surface marker CD44v9. This study aimed to quantify the effects of sulfasalazine on CD44v9 expression and pathological factors in colorectal cancers (CRCs) arising from UC. Methods: Twenty-six patients with UC-related cancer were classified into groups according to the length of sulfasalazine treatment as follows: (1) long-term (LT) (≥ 5 years) and (2) short-term (ST) (<5 years). Using immunohistochemistry, we compared CD44v9 and Ki-67 expression and pathological characteristics of each group's tumors. In vitro assay was performed to investigate the effect of sulfasalazine on epithelial differentiation and proliferation of CD44⁺ cancer cells. Results: Immunohistochemical analysis revealed that CD44v9 expression tended to be lower in the LT group (LT:ST=15.4%:46.2%, P =0.20), and Ki-67/CD44v9 double-stained cells were significantly lower in the LT group (LT:ST=0%:6.9%, P =0.01). Pathologically, the frequency of well-differentiated adenocarcinomas was higher in the LT group (LT:ST=84.6%:38.5%, P =0.04). In vitro assay revealed that sulfasalazine promoted the expression of epithelial differentiation markers (E-cadherin and CDX2) and inhibited the proliferation of CD44⁺ cancer cells. Conclusions: Long-term sulfasalazine administration reduced proliferative CD44v9⁺ cells and increased the degree of differentiation of adenocarcinomas. These findings indicate the importance of CD44v9⁺ cells in UC-related cancer progression and suggest that sulfasalazine may serve as a novel therapeutic agent that targets CD44v9⁺ cells.

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Salt-Inducible Kinase 2 Couples Ovarian Cancer Cell Metabolism with Survival at the Adipocyte-Rich Metastatic Niche

Miranda Fabrizio, Mannion David, Liu Shujuan, Zheng Yiyan, Mangala Lingegowda S., Redondo Clara, Herrero-Gonzalez Sandra, Xu Ruoyan, Taylor Charlotte, Chedom Donatien Fotso, Karaminejadranjbar Mohammad, Albukhari Ashwag, Jiang Dahai, Pradeep Sunila, Rodriguez-Aguayo Cristian, Lopez-Berestein Gabriel, Salah Eidarus, Abdul Azeez Kamal R., Elkins Jonathan M., Campo Leticia, Myers Kevin A., Klotz Daniel, Bivona Serena, Dhar Sunanda, Bast Robert C., Saya Hideyuki, Choi Hwan Geun, Gray Nathanael S., Fischer Roman, Kessler Benedikt M., Yau Christopher, Sood Anil K., Motohara Takeshi, Knapp Stefan, Ahmed Ahmed Ashour

Cancer Cell 2014.12

ISSN 1535-6108

The adipocyte-rich microenvironment forms a niche for ovarian cancer metastasis, but the mechanisms driving this process are incompletely understood. Here we show that salt-inducible kinase 2 (SIK2) is overexpressed in adipocyte-rich metastatic deposits compared with ovarian primary lesions. Overexpression of SIK2 in ovarian cancer cells promotes abdominal metastasis while . SIK2 depletion prevents metastasis in vivo. Importantly, adipocytes induce calcium-dependent activation and autophosphorylation of SIK2. Activated SIK2 plays a dual role in augmenting AMPK-induced phosphorylation of acetyl-CoA carboxylase and in activating the PI3K/AKT pathway through p85α-S154 phosphorylation. These findings identify SIK2 at the apex of the adipocyte-induced signaling cascades in cancer cells and make a compelling case for targeting SIK2 for therapy in ovarian cancer. Miranda et al. show that in serous ovarian cancer SIK2 is overexpressed in adipocyte-rich metastatic deposits compared with primary lesions. Adipocytes induce calcium-dependent activation and autophosphorylation of SIK2, which drives tumor cell survival and metabolism. SIK2 depletion prevents metastasis in vivo.

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Functional analysis of a novel glioma antigen, EFTUD1

Saito Katsuya, Iizuka Yukihiko, Ohta Shigeki, Takahashi Satoshi, Nakamura Kenta, Saya Hideyuki, Yoshida Kazunari, Kawakami Yutaka, Toda Masahiro

Neuro-Oncology 16 ( 12 ) 1618 - 1629 2014.12

ISSN 1522-8517

BACKGROUND: A cDNA library made from 2 glioma cell lines, U87MG and T98G, was screened by serological identification of antigens by recombinant cDNA expression (SEREX) using serum from a glioblastoma patient. Elongation factor Tu GTP binding domain containing protein 1 (EFTUD1), which is required for ribosome biogenesis, was identified. A cancer microarray database showed overexpression of EFTUD1 in gliomas, suggesting that EFTUD1 is a candidate molecular target for gliomas.METHODS: EFTUD1 expression in glioma cell lines and glioma tissue was assessed by Western blot, quantitative PCR, and immunohistochemistry. The effect on ribosome biogenesis, cell growth, cell cycle, and induction of apoptosis and autophagy in glioma cells during the downregulation of EFTUD1 was investigated. To reveal the role of autophagy, the autophagy-blocker, chloroquine (CQ), was used in glioma cells downregulating EFTUD1. The effect of combining CQ with EFTUD1 inhibition in glioma cells was analyzed.RESULTS: EFTUD1 expression in glioma cell lines and tissue was higher than in normal brain tissue. Downregulating EFTUD1 induced G1 cell-cycle arrest and apoptosis, leading to reduced glioma cell proliferation. The mechanism underlying this antitumor effect was impaired ribosome biogenesis via EFTUD1 inhibition. Additionally, protective autophagy was induced by glioma cells as an adaptive response to EFTUD1 inhibition. The antitumor effect induced by the combined treatment was significantly higher than that of either EFTUD1 inhibition or CQ alone.CONCLUSION: These results suggest that EFTUD1 represents a novel therapeutic target and that the combination of EFTUD1 inhibition with autophagy blockade may be effective in the treatment of gliomas.

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IGF2 preserves osteosarcoma cell survival by creating an autophagic state of dormancy that protects cells against chemotherapeutic stress

Shimizu Takatsune, Sugihara Eiji, Yamaguchi-Iwai Sayaka, Tamaki Sakura, Koyama Yuko, Kamel Walied, Ueki Arisa, Ishikawa Tomoki, Chiyoda Tatsuyuki, Osuka Satoru, Onishi Nobuyuki, Ikeda Hiroko, Kamei Junzo, Matsuo Koichi, Fukuchi Yumi, Nagai Toshihiro, Toguchida Junya, Toyama Yoshiaki, Muto Akihiro, Saya Hideyuki

Cancer Research 74 ( 22 ) 6531 - 6541 2014.11

ISSN 0008-5472

Osteosarcoma is a malignant bone tumor in children and adolescents characterized by intrinsic therapeutic resistance. The IGF2 is expressed at elevated levels in osteosarcoma after treatment with chemotherapy, prompting an examination of its functional contributions to resistance. We found that continuous exposure to IGF2 or insulin in the absence of serum created a dormant growth state in osteosarcoma cells that conferred resistance to various chemotherapeutic drugs in vitro. Mechanistic investigations revealed that this dormant state correlated with downregulation of downstream signaling by the IGF1 receptor, heightened cell survival, enhanced autophagy, and the presence of extracellular glutamine. Notably, inhibiting autophagy or depleting glutamine was sufficient to increase chemotherapeutic sensitivity in osteosarcoma xenografts in mice. Clinically, we confirmed that IGF expression levels were elevated in human osteosarcoma specimens from patients who received chemotherapy. Together, our results suggest that activation of IGF or insulin signaling preserves the survival of osteosarcoma cells under chemotherapeutic stress, providing a drug-resistant population that may engender minimal residual disease. Attenuating this survival mechanism may help overcome therapeutic resistance in osteosarcoma.

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The use of next-generation sequencing in molecular diagnosis of neurofibromatosis type 1

Maruoka Ryo, Takenouchi Toshiki, Torii Chiharu, Shimizu Atsushi, Misu Kumiko, Higasa Koichiro, Matsuda Fumihiko, Ota Arihito, Tanito Katsumi, Kuramochi Akira, Arima Yoshimi, Otsuka Fujio, Yoshida Yuichi, Moriyama Keiji, Niimura Michihito, Saya Hideyuki, Kosaki Kenjiro

Genetic Testing and Molecular Biomarkers 18 ( 11 ) 722 - 735 2014.11

AIMS: We assessed the validity of a next-generation sequencing protocol using in-solution hybridization-based enrichment to identify NF1 mutations for the diagnosis of 86 patients with a prototypic genetic syndrome, neurofibromatosis type 1. In addition, other causative genes for classic genetic syndromes were set as the target genes for coverage analysis.RESULTS: The protocol identified 30 nonsense, 19 frameshift, and 8 splice-site mutations, together with 10 nucleotide substitutions that were previously reported to be pathogenic. In the remaining 19 samples, 10 had single-exon or multiple-exon deletions detected by a multiplex ligation-dependent probe amplification method and 3 had missense mutations that were not observed in the normal Japanese SNP database and were predicted to be pathogenic. Coverage analysis of the genes other than the NF1 gene included on the same diagnostic panel indicated that the mean coverage was 115-fold, a sufficient depth for mutation detection.CONCLUSIONS: The overall mutation detection rate using the currently reported method in 86 patients who met the clinical diagnostic criteria was 92.1% (70/76) when 10 patients with large deletions were excluded. The results validate the clinical utility of this next-generation sequencing-based method for the diagnosis of neurofibromatosis type 1. Comparable detection rates can be expected for other genetic syndromes, based on the results of the coverage analysis.

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A stem cell medium containing neural stimulating factor induces a pancreatic cancer stem-like cell-enriched population

Watanabe Yusaku, Yoshimura Kiyoshi, Yoshikawa Koichi, Tsunedomi Ryoichi, Shindo Yoshitaro, Matsukuma Sou, Maeda Noriko, Kanekiyo Shinsuke, Suzuki Nobuaki, Kuramasu Atsuo, Sonoda Kouhei, Tamada Koji, Kobayashi Sei, Saya Hideyuki, Hazama Shoichi, Oka Masaki

International Journal of Oncology 45 ( 5 ) 1857 - 1866 2014.11

ISSN 1019-6439

Cancer stem cells (CSCs) have been studied for their self-renewal capacity and pluripotency, as well as their resistance to anticancer therapy and their ability to metastasize to distant organs. CSCs are difficult to study because their population is quite low in tumor specimens. To overcome this problem, we established a culture method to induce a pancreatic cancer stem-like cell (P-CSLC)-enriched population from human pancreatic cancer cell lines. Human pancreatic cancer cell lines established at our department were cultured in CSC-inducing media containing epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), neural cell survivor factor-1 (NSF-1), and N-acetylcysteine. Sphere cells were obtained and then transferred to a laminin-coated dish and cultured for approximately two months. The surface markers, gene expression, aldehyde dehydrogenase (ALDH) activity, cell cycle, and tumorigenicity of these induced cells were examined for their stem cell-like characteristics. The population of these induced cells expanded within a few months. The ratio of CD24high, CD44high, epithelial specific antigen (ESA) high, and CD44variant (CD44v) high cells in the induced cells was greatly enriched. The induced cells stayed in the G0/G1 phase and demonstrated mesenchymal and stemness properties. The induced cells had high tumorigenic potential. Thus, we established a culture method to induce a P-CSLCenriched population from human pancreatic cancer cell lines. The CSLC population was enriched approximately 100-fold with this method. Our culture method may contribute to the precise analysis of CSCs and thus support the establishment of CSC-targeting therapy.

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Loss of E-cadherin promotes migration and invasion of cholangiocarcinoma cells and serves as a potential marker of metastasis

Techasen Anchalee, Loilome Watcharin, Namwat Nisana, Khuntikeo Narong, Puapairoj Anucha, Jearanaikoon Patcharee, Saya Hideyuki, Yongvanit Puangrat

Tumor Biology 35 ( 9 ) 8645 - 8652 2014.10

ISSN 1010-4283

Tumor progression is characterized by loss of cell adhesion and increase of invasion and metastasis. E-cadherin, a cell adhesion molecule, is frequently downregulated and has been proposed as an important mediator in epithelial-mesenchymal transition (EMT) in tumors. In this study, we investigated the expression of E-cadherin and its association with cancer invasion and prognosis in cholangiocarcinoma (CCA). Immunohistochemistry results demonstrated a statistically significant association between the positive metastasis status with low E-cadherin protein expression in human CCA tissues (P = 0.04). Statistical trends were identified for low E-cadherin level and shorter survival time (P = 0.08). Targeting the E-cadherin expression in CCA cells with siRNA caused upregulation of vimentin, a mesenchymal marker, and disappearance of the E-cadherin/β-catenin adhesion complex from cell membranes. Moreover, migration and invasion abilities of the cells were increased under this condition. These findings suggest that reduction of E-cadherin contributes to CCA progression by attenuating the strength of cellular adhesion, which affects motility as well as regulating the expression of EMT-related genes during CCA invasion and metastasis. Thus, E-cadherin can act as a central modulator of tumor cell phenotype and is a potential metastasis marker in CCA.

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TNFα promotes osteosarcoma progression by maintaining tumor cells in an undifferentiated state

Mori T., Sato Y., Miyamoto K., Kobayashi T., Shimizu T., Kanagawa H., Katsuyama E., Fujie A., Hao W., Tando T., Iwasaki R., Kawana H., Morioka H., Matsumoto M., Saya H., Toyama Y., Miyamoto T.

Oncogene 33 ( 33 ) 4236 - 4241 2014.08

ISSN 0950-9232

Chronic inflammation is frequently associated with tumorigenesis in elderly people. By contrast, young people without chronic inflammation often develop tumors considered independent of chronic inflammation but driven instead by mutations. Thus, whether inflammation has a significant role in tumor progression in tumors driven by mutations remains largely unknown. Here we show that TNFα is required for the tumorigenesis of osteosarcoma, the most common tumor in children and adolescents. We show that transplantation of AX osteosarcoma cells, which harbor mutations driving c-Myc overexpression and Ink4a-deficiency, in wild-type mice promotes lethal tumorigenesis accompanied by ectopic bone formation and multiple metastases, phenotypes seen in osteosarcoma patients. Such tumorigenesis was completely abrogated in TNFα-deficient mice. AX cells have the capacity to undergo osteoblastic differentiation; however, that activity was significantly inhibited by TNFα treatment, suggesting that TNFα maintains AX cells in an undifferentiated state. TNFα inhibition of AX cell osteoblastic differentiation occurred through ERK activation, and a pharmacological TNFα inhibitor effectively inhibited both AX cell tumorigenesis and increased osteoblastic gene expression and increased survival of tumor-bearing mice. Lethal tumorigenesis of AX cells was also abrogated in IL-1α/IL-1β doubly deficient mice. We found that both TNFα and IL-1 maintained AX cells in an undifferentiated state via ERK activation. Thus, inflammatory cytokines are required to promote tumorigenesis even in mutation-induced tumors, and TNFα/IL-1 and ERK may represent therapeutic targets for osteosarcoma.

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TNF-/TNFR1 signaling promotes gastric tumorigenesis through induction of Noxo1 and Gna14 in tumor cells

Oshima H., Ishikawa T., Yoshida G. J., Naoi K., Maeda Y., Naka K., Ju X., Yamada Y., Minamoto T., Mukaida N., Saya H., Oshima M.

Oncogene 33 ( 29 ) 3820 - 3829 2014.07

ISSN 0950-9232

Helicobacter pylori infection induces chronic inflammation that contributes to gastric tumorigenesis. Tumor necrosis factor (TNF-) is a proinflammatory cytokine, and polymorphism in the TNF- gene increases the risk of gastric cancer. We herein investigated the role of TNF- in gastric tumorigenesis using Gan mouse model, which recapitulates human gastric cancer development. We crossed Gan mice with TNF- (Tnf) or TNF- receptor TNFR1 (Tnfrsf1a) knockout mice to generate Tnf-/-Gan and Tnfrsf1a-/-Gan mice, respectively, and examined their tumor phenotypes. Notably, both Tnf-/-Gan mice and Tnfrsf1a-/-Gan mice showed similar, significant suppression of gastric tumor growth compared with control Tnf+/+ or Tnfrsf1a+/+ Gan mice. These results indicate that TNF- signaling through TNFR1 is important for gastric tumor development. Bone marrow (BM) transplantation experiments showed that TNF- expressed by BM-derived cells (BMDCs) stimulates the TNFR1 on BMDCs by an autocrine or paracrine manner, which is important for gastric tumor promotion. Moreover, the microarray analysis and colony formation assay indicated that NADPH oxidase organizer 1 (Noxo1) and Gna14 are induced in tumor epithelial cells in a TNF-dependent manner, and have an important role in tumorigenicity and tumor-initiating cell property of gastric cancer cells. Accordingly, it is possible that the activation of TNF-/TNFR1 signaling in the tumor microenvironment promotes gastric tumor development through induction of Noxo1 and Gna14, which contribute to maintaining the tumor cells in an undifferentiated state. The present results indicate that targeting the TNF-/TNFR1 pathway may be an effective preventive or therapeutic strategy for gastric cancer.

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Effectiveness of plasma treatment on gastric cancer cells

Torii Koji, Yamada Suguru, Nakamura Kae, Tanaka Hiromasa, Kajiyama Hiroaki, Tanahashi Kuniaki, Iwata Naoki, Kanda Mitsuro, Kobayashi Daisuke, Tanaka Chie, Fujii Tsutomu, Nakayama Goro, Koike Masahiko, Sugimoto Hiroyuki, Nomoto Shuji, Natsume Atsushi, Fujiwara Michitaka, Mizuno Masaaki, Hori Masaru, Saya Hideyuki, Kodera Yasuhiro

Gastric Cancer 18 ( 3 ) 635 - 643 2014.07

ISSN 1436-3291

Background: Treatment of peritoneal carcinomatosis arising from gastric cancer remains a considerable challenge. In recent years, the anticancer effect of nonequilibrium atmospheric pressure plasma (NEAPP) has been reported in several cancer cell lines. Use of NEAPP may develop into a new class of anticancer therapy that augments surgery, chemotherapy, and radiotherapy. Method: Gastric cancer cells were assessed for changes in cell morphology and rate of proliferation after treatment with NEAPP-exposed medium (PAM). To explore the functional mechanism, caspase 3/7, annexin V, and uptake of reactive oxygen species (ROS) were evaluated, along with the effect of the ROS scavenger N-acetylcysteine (NAC). Results: PAM treatment for 24 h affected cell morphology, suggestive of induction of apoptosis. PAM cytotoxicity was influenced by the time of exposure to PAM, the type of cell line, and the number of cells seeded. Cells treated with PAM for 2 h demonstrated activated caspase 3/7 and an increased proportion of annexin V-positive cells compared with untreated cells. Additionally, ROS uptake was observed in PAM-treated cells, whereas NAC reduced the cytotoxicity induced by PAM presumably through reduction of ROS uptake. Furthermore, CD44 variant 9, which reportedly leads to glutathione synthesis and suppresses stress signaling of ROS, was overexpressed in PAM-resistant cells. Conclusions: PAM treatment induced apoptosis of gastric cancer cells through generation and uptake of ROS. Local administration of PAM could develop into an option to treat peritoneal carcinomatosis.

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TSPAN2 Is Involved in Cell Invasion and Motility during Lung Cancer Progression

Otsubo Chihiro, Otomo Ryo, Miyazaki Makoto, Matsushima-Hibiya Yuko, Kohno Takashi, Iwakawa Reika, Takeshita Fumitaka, Okayama Hirokazu, Ichikawa Hitoshi, Saya Hideyuki, Kiyono Tohru, Ochiya Takahiro, Tashiro Fumio, Nakagama Hitoshi, Yokota Jun, Enari Masato

Cell Reports 7 ( 2 ) 527 - 538 2014.04

In lung cancer progression, p53 mutations are more often observed in invasive tumors than in noninvasive tumors, suggesting that p53 is involved in tumor invasion and metastasis. To understand the nature of p53 function as a tumor suppressor, it is crucial to elucidate the detailed mechanism of the alteration in epithelial cells that follow oncogenic KRAS activation and p53 inactivation. Here, we report that KRAS activation induces epithelial-mesenchymal transitionand that p53 inactivation is required for cell motility and invasiveness. Furthermore, TSPAN2, atransmembrane protein, is responsible for cell motility and invasiveness elicited by p53 inactivation. TSPAN2 is highly expressed in p53-mutated lung cancer cells, and high expression of TSPAN2 is associated with the poor prognosis of lung adenocarinomas. TSPAN2 knockdown suppresses metastasis tothe lungs and liver, enabling prolonged survival. TSPAN2 enhances cell motility and invasiveness by assisting CD44 in scavenging intracellular reactive oxygen species.

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Targeting Cancer Stem Cells-Modulating Embryonic Stem Cell Signaling, Epigenetics, and Tumor Metabolism

Takebe Naoko, Harris Pamela Jo, Kondo Yutaka, Nair Abhilasha, Percy Ivy S., Saya Hideyuki

295 - 317 2014.04

Cancer stem cells (CSC) are important to understand in order to develop solutions to treat tumor metastasis and resistance to therapy. CSCs are characterized by their ability to self-renew and differentiate into tumors, ultimately leading to therapeutic resistance. This chapter summarizes the various potential therapeutic approaches to control CSCs survival and proliferation based on preclinical and clinical study-based rationale. The proposed agents range from investigational, which are still in pre-clinical stage, already in early clinical investigational stage, or already approved for clinical use for other indications. The manuscript covers the agents targeting embryonic stem cell signaling, epigenetic control, cancer metabolism, and induction of epithelial-to-mesenchymal transition (EMT). The combination of therapeutic strategies to reduce bulk tumor populations and to target CSCs population may provide clinically meaningful tumor suppression. The development of new anti-CSC therapeutic agents and the complexity of potential molecular pathways controlling CSC survival are described in this review.

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Glycan profiling of gestational choriocarcinoma using a lectin microarray

Kobayashi Yusuke, Masuda Kenta, Banno Kouji, Kobayashi Nana, Umene Kiyoko, Nogami Yuya, Tsuji Kosuke, Ueki Arisa, Nomura Hiroyuki, Sato Kenji, Tominaga Eiichiro, Shimizu Takatsune, Saya Hideyuki, Aoki Daisuke

Oncology Reports 31 ( 3 ) 1121 - 1126 2014.03

ISSN 1021-335X

Glycosylation is an important post-translational modification, in which attachment of glycans to proteins has effects on biological functions and carcinogenesis. Analysis of human chorionic gonadotropin, a glycoprotein hormone produced by placental trophoblasts and trophoblastic tumors, has contributed to the diagnosis and treatment of trophoblastic disease, resulting in reduced incidence and mortality. However, alterations of the glycan structure itself in choriocarcinoma have not been characterized. We established a new choriocarcinoma cell line, induced choriocarcinoma cell-1 (iC³-1), which mimics the clinical pathohistology in vivo, to examine the tumorigenesis and pathogenesis of choriocarcinoma. In this study, the alterations of glycan structures in the development of choriocarcinoma were examined by performance of comprehensive glycan profiling in clinical samples and in iC³-1 cells using a conventional microarray and the recently introduced lectin microarray. Microarray comparison showed significant upregulation of several characteristic glycogenes in the iC³-1 cells as compared to the parental HTR8/SVneo cells. The lectin array showed increased α-2-6-sialic acid, Galβ1-4GlcNAc, GlcNAcβ1-3GalNAc, and decreased α-1-6 core fucose, high mannose, GalNacβ1-4Gal, GALNAc (Tn antigen) and Galβ-3Gal in choriocarcinoma tissue compared to normal villi. This is the first report of a lectin array analysis in choriocarcinoma and provides useful information for understanding of the disease.

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CD44s signals the acquisition of the mesenchymal phenotype required for anchorage-independent cell survival in hepatocellular carcinoma

Okabe H., Ishimoto T., Mima K., Nakagawa S., Hayashi H., Kuroki H., Imai K., Nitta H., Saito S., Hashimoto D., Chikamoto A., Ishiko T., Watanabe M., Nagano O., Beppu T., Saya H., Baba H.

British Journal of Cancer 110 ( 4 ) 958 - 966 2014.02

ISSN 0007-0920

Background:Circulating tumour cells (CTCs) have an important role in metastatic processes, but details of their basic characteristics remain elusive. We hypothesised that CD44-expressing CTCs show a mesenchymal phenotype and high potential for survival in hepatocellular carcinoma (HCC).Methods:Circulating CD44 + CD90 + cells, previously shown to be tumour-initiating cells, were sorted from human blood and their genetic characteristics were compared with those of tumour cells from primary tissues. The mechanism underlying the high survival potential of CD44-expressing cells in the circulatory system was investigated in vitro.Results:CD44 + CD90 + cells in the blood acquired epithelial-mesenchymal transition, and CD44 expression remarkably increased from the tissue to the blood. In Li7 and HLE cells, the CD44 high population showed higher anoikis resistance and sphere-forming ability than did the CD44 low population. This difference was found to be attributed to the upregulation of Twist1 and Akt signal in the CD44 high population. Twist1 knockdown showed remarkable reduction in anoikis resistance, sphere formation, and Akt signal in HLE cells. In addition, mesenchymal markers and CD44s expression were downregulated in the Twist1 knockdown.Conclusions:CD44s symbolises the acquisition of a mesenchymal phenotype regulating anchorage-independent capacity. CD44s-expressing tumour cells in peripheral blood are clinically important therapeutic targets in HCC.

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Multiple café au lait spots in familial patients with MAP2K2 mutation

Takenouchi Toshiki, Shimizu Atsushi, Torii Chiharu, Kosaki Rika, Takahashi Takao, Saya Hideyuki, Kosaki Kenjiro

American Journal of Medical Genetics, Part A 164 ( 2 ) 392 - 396 2014.02

ISSN 1552-4825

Recent advances in genetic diagnostic technologies have made the classic disease nosology highly complicated. This situation is exemplified by rasopathies, among which neurofibromatosis type 1 and Noonan syndrome represent prototypic entities. The former condition is characterized by multiple café au lait spots and neurofibromas, while the latter is characterized by distinct facial features, webbed neck, congenital heart disease, and a short stature. On rare occasions, the features of both neurofibromatosis and Noonan syndrome co-exist within an individual; such patients are diagnosed as having neurofibromatosis-Noonan syndrome. Here, we report familial patients with multiple café au lait spots and Noonan syndrome-like facial features. A mutation analysis unexpectedly revealed a mutation in MAP2K2 in both the propositus and his mother. The propositus fulfilled the diagnostic criteria for neurofibromatosis type 1, but his mother did not. Their phenotype was not consistent with that of cardio-facio-cutaneous syndrome, which is classically known to be associated with MAP2K2 mutations. The mother of the propositus had cervical cancer at the age of 23 years, consistent with the oncogenic tendency associated with rasopathies. The phenotypic combination of multiple café au lait spots and Noonan syndrome-like facial features suggested a diagnosis of neurofibromatosis-Noonan syndrome. Whether this condition represents a discrete disease entity or a variable expression of neurofibromatosis type 1 has long been debated. The present observation suggests that some perturbation in the RAS/MAPK signaling cascade results in multiple café au lait spots, a key diagnostic phenotype of rasopathies, although the exact mechanism remains to be elucidated.

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Ink4a/Arf^-/- and HRAS(G12V) transform mouse mammary cells into triple-negative breast cancer containing tumorigenic CD49f^- quiescent cells

Kai K., Iwamoto T., Kobayashi T., Arima Y., Takamoto Y., Ohnishi N., Bartholomeusz C., Horii R., Akiyama F., Hortobagyi G. N., Pusztai L., Saya H., Ueno N. T.

Oncogene 33 ( 4 ) 440 - 448 2014.01

ISSN 0950-9232

Intratumoral heterogeneity within individual breast tumors is a well-known phenomenon that may contribute to drug resistance. This heterogeneity is dependent on several factors, such as types of oncogenic drivers and tumor precursor cells. The purpose of our study was to engineer a mouse mammary tumor model with intratumoral heterogeneity by using defined genetic perturbations. To achieve this, we used mice with knockout (-/-) of Ink4a/Arf, a tumor suppressor locus; these mice are known to be susceptible to non-mammary tumors such as fibrosarcoma. To induce mammary tumors, we retrovirally introduced an oncogene, HRAS(G12V), into Ink4a/Arf^-/- mammary cells in vitro, and those cells were inoculated into syngeneic mice mammary fat pads. We observed 100% tumorigenesis. The tumors formed were negative for estrogen receptor, progesterone receptor and HER2. Further, they had pathological features similar to those of human triple-negative breast cancer (TNBC) (for example, pushing borders, central necrosis). The tumors were found to be heterogeneous and included two subpopulations: CD49f^- quiescent cells and CD49f ⁺ cells. Contrary to our expectation, CD49f^- quiescent cells had high tumor-initiating potential and CD49f⁺ cells had relatively low tumor-initiating potential. Gene expression analysis revealed that CD49f^- quiescent cells overexpressed epithelial-to-mesenchymal transition-driving genes, reminiscent of tumor-initiating cells and claudin-low breast cancer. Our animal model with intratumoral heterogeneity, derived from defined genetic perturbations, allows us to test novel molecular targeted drugs in a setting that mimics the intratumoral heterogeneity of human TNBC.

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Inversed relationship between CD44 variant and c-Myc due to oxidative stress-induced canonical Wnt activation

Yoshida Go J., Saya Hideyuki

Biochemical and Biophysical Research Communications 443 ( 2 ) 622 - 627 2014.01

ISSN 0006-291X

Cancer stem-like cells express high amount of CD44 variant8-10 which protects cancer cells from redox stress. We have demonstrated by immunohistochemical analysis and Western blotting, and reverse-transcription polymerase chain reaction, that CD44 variant8-10 and c-Myc tend to show the inversed expression manner in gastric cancer cells. That is attributable to the oxidative stress-induced canonical Wnt activation, and furthermore, the up-regulation of the downstream molecules, one of which is oncogenic c-Myc, is not easily to occur in CD44 variant-positive cancer cells. We have also found out that CD44v8-10 expression is associated with the turn-over of the c-Myc with the experiments using gastric cancer cell lines. This cannot be simply explained by the model of oxidative stress-induced Wnt activation. CD44v8-10-positive cancer cells are enriched at the invasive front. Tumor tissue at the invasive area is considered to be composed of heterogeneous cellular population; dormant cancer stem-like cells with CD44v8-10 ^high/ Fbw7 ^high/ c-Myc ^low and proliferative cancer stem-like cells with CD44v8-10 ^high/ Fbw7 ^low/ c-Myc ^high.

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EpCAM expression in the prostate cancer makes the difference in the response to growth factors

Yoshida Go J., Saya Hideyuki

Biochemical and Biophysical Research Communications 443 ( 1 ) 239 - 245 2014.01

ISSN 0006-291X

Introduction: Epithelial cell adhesion molecule (EpCAM) is expressed in tumors with an epithelial cell of origin, in a heterogeneous manner. Prostate cancer stem-like cells highly express EpCAM. However, little is known about how EpCAM is involved in the ability of cells to adapt to micro-environmental changes in available growth factors, which is one of the essential biological phenotypes of cancer stem-like cells (CSCs). Methods: EpCAM-high and EpCAM-low subpopulations of cells were established from the prostate cancer cell line PC-3. Signal transductions in response to serum starvation, and on the exposure to EGF ligand or the specific inhibitor were analyzed in terms. Furthermore, we analyzed the expression level of amino acid transporters which contribute to the activation of mTOR signal between the two subgroups. Results: EpCAM-high and EpCAM-low PC-3 subpopulations showed markedly different responses to serum starvation. EpCAM expression was positively correlated with activation of the mTOR and epithelial growth factor receptor (EGFR) signaling pathways. Furthermore, AMP-activated protein kinase (AMPK) was gradually de-activated in EpCAM-low PC-3 cells in the absence of serum. Conclusions: EpCAM regulates the AMPK signaling pathway, essential for the response to growth factors characterized by EGF. LAT1, the amino acid transporter stabilized at the cellular membrane by EpCAM, is likely to be responsible for the difference in the susceptibility to EGF between EpCAM-high and EpCAM-low PC-3 cells.

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Expression of CD24 is associated with HER2 expression and supports HER2-Akt signaling in HER2-positive breast cancer cells

Hosonaga Mari, Arima Yoshimi, Sugihara Eiji, Kohno Norio, Saya Hideyuki

Cancer Science 105 ( 7 ) 779 - 787 2014

ISSN 1347-9032

Human epidermal growth factor receptor 2 (HER2)-positive breast cancer is treated with HER2-targeted agents, such as trastuzumab and lapatinib, that suppress signaling by phosphatidylinositol 3-kinase (PI3K)-Akt and MAPK pathways. However, resistance to HER2-targeted therapy remains a major clinical problem. Overexpression of CD24 has been detected in many cancers and is associated with a poor prognosis in women with breast cancer. HER2-positive breast tumors are predominantly positive for CD24, suggesting that the expression of the two molecules is related. To investigate the relation between HER2 and CD24, we overexpressed HER2 in breast cancer cells that were triple-negative for the estrogen receptor, progesterone receptor and HER2. We found that expression of CD24 was increased by stable overexpression of HER2. Flow cytometry thus revealed that the percentage of CD24-positive cells was markedly higher in the HER2-positive fraction than in the HER2-negative fraction. Knockdown of CD24 in breast cancer cells positive for endogenous HER2 downregulated HER2 expression, whereas knockdown of HER2 did not affect the expression of CD24. Knockdown of CD24 also suppressed the phosphorylation of Akt, which functions downstream of HER2 and PI3K to promote cell survival. Moreover, knockdown of CD24 increased the sensitivity of HER2-positive breast cancer cells to lapatinib treatment. Our results thus indicate that CD24 supports both the expression of HER2 and the consequent activation of PI3K-Akt signaling. Furthermore, CD24 may contribute to resistance to HER2-targeted therapy and, therefore, is itself a potential therapeutic target in HER2-positive breast cancer. The knockdown of CD24 decreased the phosphorylation of Akt in HER2-positive breast cancer cells. These results suggest that CD24 supports HER2-Akt signaling pathway.

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Macrophage-derived reactive oxygen species suppress miR-328 targeting CD44 in cancer cells and promote redox adaptation

Ishimoto Takatsugu, Sugihara Hidetaka, Watanabe Masayuki, Sawayama Hiroshi, Iwatsuki Masaaki, Baba Yoshifumi, Okabe Hirohisa, Hidaka Kosei, Yokoyama Naomi, Miyake Keisuke, Yoshikawa Momoko, Nagano Osamu, Komohara Yoshihiro, Takeya Motohiro, Saya Hideyuki, Baba Hideo

Carcinogenesis 35 ( 5 ) 1003 - 1011 2014

ISSN 0143-3334

CD44 is frequently overexpressed in a wide variety of epithelial malignancies including gastrointestinal cancer and causes resistance to currently available treatments. MicroRNAs (miRNAs) are non-coding RNAs that regulate molecular pathways in cancer by targeting various genes. The aim of this study was to investigate the regulation of CD44 expression by miRNAs and to develop new molecular targets in gastrointestinal cancer. We performed miRNA screening in six human gastrointestinal cancer cell lines and identified three candidate miRNAs that could regulate CD44 expression in gastrointestinal cancer. Among these, we focused on miR-328 and examined its functional relevance using growth assays and cytotoxicity assays. CD44 expression was reduced in gastrointestinal cancer cell lines forced to express miR-328, leading to inhibition of cancer cell growth in vitro and in vivo, and impaired resistance to chemotherapeutic drugs and reactive oxygen species (ROS). In contrast, induction of CD44 expression by miR-328 inhibitor led to promotion of cancer cell growth. Furthermore, we revealed that ROS produced by macrophages triggered CD44 expression through suppression of miR-328 in gastric cancer cells. Finally, tumor-infiltrating macrophages (CD68 and CD163) were closely related to both miR-328 downregulation and CD44 upregulation in 63 patients with surgically resected gastric cancer. These findings suggest that macrophages in the tumor microenvironment may cause increased CD44 expression through miR-328 suppression, resulting in tumor progression by enhancing ROS defense. miR-328-CD44 signaling mediated by macrophages may thus represent a potential target for the treatment of gastrointestinal cancer.

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Regulation of MKL1 via actin cytoskeleton dynamics drives adipocyte differentiation

Nobusue Hiroyuki, Onishi Nobuyuki, Shimizu Takatsune, Sugihara Eiji, Oki Yoshinao, Sumikawa Yuko, Chiyoda Tatsuyuki, Akashi Koichi, Saya Hideyuki, Kano Koichiro

Nature Communications 5 3368 2014

Cellular differentiation is regulated through activation and repression of defined transcription factors. A hallmark of differentiation is a pronounced change in cell shape, which is determined by dynamics of the actin cytoskeleton. Here we show that regulation of the transcriptional coactivator MKL1 (megakaryoblastic leukemia 1) by actin cytoskeleton dynamics drives adipocyte differentiation mediated by peroxisome proliferator-activated receptor γ (PPARγ), a master transcriptional regulator of adipogenesis. Induction of adipocyte differentiation results in disruption of actin stress fibres through downregulation of RhoA-ROCK signalling. The consequent rapid increase in monomeric G-actin leads to the interaction of G-actin with MKL1, which prevents nuclear translocation of MKL1 and allows expression of PPARγ followed by adipogenic differentiation. Moreover, we found that MKL1 and PPARγ act in a mutually antagonistic manner in the adipocytic differentiation programme. Our findings thus provide new mechanistic insight into the relation between the dynamics of cell shape and transcriptional regulation during cellular differentiation.

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Basic biology of gliomas

Saya Hideyuki, Shibao Shunsuke, Sampetrean Oltea

Japanese Journal of Neurosurgery 23 ( 7 ) 541 - 546 2014

ISSN 0917-950X

Gliomas are a group of primary brain neoplasms classified mainly on the basis of their morphological resemblance to glial cells. Their most malignant form, grade IV glioma or glioblastoma multiforme, continues to remain lethal, partly due to a pronounced intratumoral heterogeneity which prevents effective therapeutic targeting. The heterogeneity seen in glioblastomas ranges from multiple genetic and epigenetic alterations to a cellular diversity that has recently been traced to the existence of tumor stem cells.Glioma stem cells, like other types of cancer stem cells, have the ability to self-renew and differentiate into multiple tissue-specific lineages. They can therefore maintain and propagate the tumor by continuously giving rise to a variety of malignant offspring and also initiate tumor recurrence from a very small number of cells. Furthermore, they are also considered to be the cells most likely to survive even multi-modality treatments, due to characteristics such as slow cycling or quiescence, high drug efflux capacity and strong antioxidant and immune defense systems.With the addition of each new research finding, the concept of glioma stem cell itself continues to evolve. However, many aspects of glioma stem cell biology are still insufficiently understood. It is not known which cells they originate from, what their relationship to normal tissue stem cells is and what mechanisms or events underlie their malignant transformation. Furthermore, whether the answers to these questions are the same for all glioma stem cells is also a matter that requires further investigation.This review focuses on our current understanding of glioma stem cells, the signaling pathways behind their pathogenic features and their role in glioma biology. We also discuss possible answers to the questions mentioned above, in the context of knowledge obtained from the study of tumor stem cells of several other types of malignancies. Finally, we review the importance of glioma stem cells in treatment resistance in glioblastoma and the implications of stem cell-specific targeting strategies.

A human cancer xenograft model utilizing normal pancreatic duct epithelial cells conditionally transformed with defined oncogenes

Inagawa Yuki, Yamada Kenji, Yugawa Takashi, Ohno Shin Ichi, Hiraoka Nobuyoshi, Esaki Minoru, Shibata Tatsuhiro, Aoki Kazunori, Saya Hideyuki, Kiyono Tohru

Carcinogenesis 35 ( 8 ) 2014

ISSN 0143-3334

Pancreatic ductal adenocarcinomas (PDACs) are considered to arise through neoplastic transformation of human pancreatic duct epithelial cells (HPDECs). In order to evaluate the biological significance of genetic and epigenetic alterations in PDACs, we isolated primary HPDECs and established an in vitro carcinogenesis model. Firstly, lentivirus-mediated transduction of KRAS^G12V, MYC and human papillomavirus 16 (HPV16) E6/E7 under the control of a tetracyclin-inducible promoter efficiently immortalized and transformed primary HPDECs, which gave rise to adenocarcinomas subcutaneously in an immune-deficient mouse xenograft model, depending on expression of the four genes. The tumors regressed promptly upon shutting-off the oncogenes, and the remaining tissues showed histological features corresponding to normal ductal structures with simple columnar epithelium. Re-expression of the oncogenes resulted in development of multiple PDACs through pancreatic intraepithelial neoplasia-like structures. We also succeeded in efficient immortalization of primary HPDECs with transduction of mutant CDK4, cyclin D1 and TERT. The cells maintained a normal diploid status and formed duct-like structures in a three dimensional culture. In combination with p53 silencing, KRAS^G12V alone was sufficient to fully transform the immortalized HPDECs, and MYC markedly accelerated the development of tumors. Our PDAC model supports critical roles of KRAS mutations, inactivation of the p53 and p16-pRB pathways, active telomerase and MYC expression in pancreatic carcinogenesis and thus recapitulates many features of human PDAC development. The present system with reversible control of oncogene expression enabled de novo development of PDAC from quasi-normal human tissues pre-formed subcutaneously in mice and might be applicable to carcinogenesis models in many organ sites.

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Tumor necrosis factor-α modulates epithelial mesenchymal transition mediators ZEB2 and S100A4 to promote cholangiocarcinoma progression

Techasen Anchalee, Namwat Nisana, Loilome Watcharin, Duangkumpha Kassaporn, Puapairoj Anucha, Saya Hideyuki, Yongvanit Puangrat

Journal of Hepato-Biliary-Pancreatic Sciences 21 ( 9 ) 703 - 711 2014

ISSN 1868-6974

Background The epithelial-mesenchymal transition (EMT) process strongly contributes to cancer metastasis. This study was to investigate the alteration of EMT-related proteins (ZEB1, ZEB2 and S100A4) in cholangiocarcinoma (CCA) tissues. The effect of tumor necrosis factor-α (TNF-α) on the expression of those molecules in CCA cells was investigated. Methods The quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was used to quantify ZEB1, ZEB2 and S100A4 mRNA levels in 50 CCA tissues and related its expression to clinicopathological data. ZEB2 protein immunostaining was investigated in 165 CCA tissues. The effect of TNF-α on EMT-related CCA cell migration was evaluated using qRT-PCR, immunofluorescence and transwell migration assays. Results ZEB2 and S100A4 mRNA levels were found to be higher in CCA tissues. High levels of S100A4 mRNA and ZEB2 protein were significantly associated with CCA metastasis (P = 0.04 and P = 0.03). Moreover, a trend toward statistical association was found with high levels of both ZEB2 mRNA and protein with shorter survival time (P = 0.10 and P = 0.19). In addition, TNF-α induced CCA cell migration by the induction of transforming growth factor-β (TGF-β) resulting in ZEB2 and S100A4 mRNA and protein activation. Conclusions These studies demonstrate that TNF-α plays crucial role in the progression of CCA by activating TGF-β signaling and the induction of ZEB2 and S100A4, EMT-related proteins expression.

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Integrated analysis identifies different metabolic signatures for tumor-initiating cells in a murine glioblastoma model

Saga Isako, Shibao Shunsuke, Okubo Jun, Osuka Satoru, Kobayashi Yusuke, Yamada Sachiko, Fujita Satoshi, Urakami Kenichi, Kusuhara Masatoshi, Yoshida Kazunari, Saya Hideyuki, Sampetrean Oltea

Neuro-Oncology 16 ( 8 ) 1048 - 1056 2014

ISSN 1522-8517

Background The metabolic preference of malignant glioma for glycolysis as an energy source is a potential therapeutic target. As a result of the cellular heterogeneity of these tumors, however, the relation between glycolytic preference, tumor formation, and tumor cell clonogenicity has remained unknown. To address this issue, we analyzed the metabolic profiles of isogenic glioma-initiating cells (GICs) in a mouse model. Methods GICs were established by overexpression of H-RasV12 in Ink4a/Arf-null neural stem cells. Subpopulations of these cells were obtained by single-cell cloning, and clones differing in extracellular acidification potential were assessed for metabolic characteristics. Tumors formed after intracranial implantation of these clones in mice were examined for pathological features of glioma and expression of glycolytic enzymes. Results Malignant transformation of neural stem cells resulted in a shift in metabolism characterized by an increase in lactic acid production. However, isogenic clonal populations of GICs manifested pronounced differences in glucose and oxygen consumption, lactate production, and nucleoside levels. These differences were paralleled by differential expression of glycolytic enzymes such as hexokinase 2 and pyruvate kinase M2, with this differential expression also being evident in tumors formed by these clones in vivo. Conclusions The metabolic characteristics of glioma cells appear early during malignant transformation and persist until the late stages of tumor formation. Even isogenic clones may be heterogeneous in terms of metabolic features, however, suggesting that a more detailed understanding of the metabolic profile of glioma is imperative for effective therapeutic targeting.

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Katanin p60 contributes to microtubule instability around the midbody and facilitates cytokinesis in rat cells

Matsuo Moe, Shimodaira Tetsuhiro, Kasama Takashi, Hata Yukie, Echigo Ayumi, Okabe Masaki, Arai Kazuya, Makino Yasutaka, Niwa Shin Ichiro, Saya Hideyuki, Kishimoto Toshihiko

PLoS One 8 ( 11 ) 2013.11

The completion of cytokinesis is crucial for mitotic cell division. Cleavage furrow ingression is followed by the breaking and resealing of the intercellular bridge, but the detailed mechanism underlying this phenomenon remains unknown. Katanin is a microtubule-severing protein comprised of an AAA ATPase subunit and an accessory subunit designated as p60 and p80, respectively. Localization of katanin p60 was observed at the midzone to midbody from anaphase to cytokinesis in rat cells, and showed a ring-shaped distribution in the gap between the inside of the contractile ring and the central spindle bundle in telophase. Katanin p60 did not bind with p80 at the midzone or midbody, and localization was shown to be dependent on microtubules. At the central spindle and the midbody, no microtubule growth plus termini were seen with katanin p60, and microtubule density was inversely correlated with katanin p60 density in the region of katanin p60 localization that seemed to lead to microtubule destabilization at the midbody. Inhibition of katanin p60 resulted in incomplete cytokinesis by regression and thus caused the appearance of binucleate cells. These results suggest that katanin p60 contributes to microtubule instability at the midzone and midbody and facilitates cytokinesis in rat cells.

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Redox regulation in stem-like cancer cells by CD44 variant isoforms

Nagano O., Okazaki S., Saya H.

Oncogene 32 ( 44 ) 5191 - 5198 2013.10

ISSN 0950-9232

Increasing evidence indicates that several types of solid tumor are hierarchically organized and sustained by a distinct population of cancer stem cells (CSCs). CSCs possess enhanced mechanisms of protection from stress induced by reactive oxygen species (ROS) that render them resistant to chemo-and radiotherapy. Expression of CD44, especially variant isoforms (CD44v) of this major CSC marker, contributes to ROS defense through upregulation of the synthesis of reduced glutathione (GSH), the primary intracellular antioxidant. CD44v interacts with and stabilizes xCT, a subunit of the cystine-glutamate transporter xc(-), and thereby promotes cystine uptake for GSH synthesis. Given that cancer cells are often exposed to high levels of ROS during tumor progression, the ability to avoid the consequences of such exposure is required for cancer cell survival and propagation in vivo. CSCs, in which defense against ROS is enhanced by CD44v are thus thought to drive tumor growth, chemoresistance and metastasis. Therapy targeted to the CD44v-xCT system may therefore impair the ROS defense ability of CSCs and thereby sensitize them to currently available treatments.

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Characteristics of glioma stem cells

Sampetrean Oltea, Saya Hideyuki

Brain Tumor Pathology 30 ( 4 ) 209 - 214 2013.10

ISSN 1433-7398

The cancer stem cell theory postulates that tumors are sustained by a select cell population with specific features, such as self-renewal ability and the capacity to give rise to a heterogeneous mass of tumor cells. The existence of such cells has been demonstrated for glioblastoma, with these cells being referred to as glioma stem cells (GSCs). Glioblastomas are notoriously heterogeneous tumors, however, and the isolation and characterization of their stem cells will require further investigations. Furthermore, the lack of unequivocal markers for GSCs and a partial overlap in characteristics with other cells often lead to confusion. Here, we review the characteristics necessary for a glioma cell to be considered a stem cell, and we adopt our murine glioblastoma model based on genetically modified neural stem cells to illustrate and discuss the GSC concept.

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Functional role of CD44v-xCT system in the development of spasmolytic polypeptide-expressing metaplasia

Wada Takeyuki, Ishimoto Takatsugu, Seishima Ryo, Tsuchihashi Kenji, Yoshikawa Momoko, Oshima Hiroko, Oshima Masanobu, Masuko Takashi, Wright Nicholas A., Furuhashi Satoshi, Hirashima Kotaro, Baba Hideo, Kitagawa Yuko, Saya Hideyuki, Nagano Osamu

Cancer Science 104 ( 10 ) 1323 - 1329 2013.10

ISSN 1347-9032

Cancer development is often preceded by the appearance of preneoplastic lesions. In gastric carcinogenesis, chronic inflammation and histopathologic progression of the stomach epithelium lead to the development of metaplasia and eventually adenocarcinoma. The cell surface protein CD44, especially its variant isoforms (CD44v), has been implicated in metaplasia-carcinoma sequence progression in the stomach. We recently found that CD44v interacts with and stabilizes xCT, a subunit of the cystine transporter system xc(-), in cancer cells and thereby increases cystine uptake and confers resistance to various types of cellular stress in vivo. The functional relevance of CD44v and xCT in the development of preneoplastic lesions, however, has remained unknown. We have now examined the role of the CD44v-xCT system in the development of spasmolytic polypeptide-expressing metaplasia (SPEM) in mouse models of gastric carcinogenesis. CD44v was found to be expressed de novo in SPEM, and CD44v⁺ metaplastic cells manifested upregulation of xCT expression compared with CD44v^- cells. Genetic ablation of CD44 or treatment with sulfasalazine, an inhibitor of xCT-dependent cystine transport, suppressed the development of SPEM and subsequent gastric tumor growth. Therapy targeted to CD44v-xCT could thus prove effective for prevention or attenuation of the CD44v-dependent development of preneoplastic lesions and cancer.

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Statins Decrease Lung Inflammation in Mice by Upregulating Tetraspanin CD9 in Macrophages

Jin Yingji, Tachibana Isao, Takeda Yoshito, He Ping, Kang Sujin, Suzuki Mayumi, Kuhara Hanako, Tetsumoto Satoshi, Tsujino Kazuyuki, Minami Toshiyuki, Iwasaki Takeo, Nakanishi Kaori, Kohmo Satoshi, Hirata Haruhiko, Takahashi Ryo, Inoue Koji, Nagatomo Izumi, Kida Hiroshi, Kijima Takashi, Ito Mari, Saya Hideyuki, Kumanogoh Atsushi

PLoS One 8 ( 9 ) 2013.09

Tetraspanins organize protein complexes in tetraspanin-enriched membrane microdomains that are distinct from lipid rafts. Our previous studies suggested that reduction in the levels of tetraspanins CD9 and CD81 may be involved in the progression of inflammatory lung diseases, especially COPD. To search for agents that increase the levels of these tetraspanins, we screened 1,165 drugs in clinical use and found that statins upregulate CD9 and CD81 in RAW264.7 macrophages. The lipophilic statins, fluvastatin and simvastatin, reversed LPS-induced downregulation of CD9 and CD81, simultaneously preventing TNF-α and matrix metalloproteinase-9 production and spreading of RAW264.7 cells. These statins exerted anti-inflammatory effects in vitro in wild-type macrophages but not in CD9 knockout macrophages, and decreased lung inflammation in vivo in wild-type mice but not in CD9 knockout mice, suggesting that their effects are dependent on CD9. Mechanistically, the statins promoted reverse transfer of the LPS-signaling mediator CD14 from lipid rafts into CD9-enriched microdomains, thereby preventing LPS receptor formation. Finally, upregulation of CD9/CD81 by statins was related to blockade of GTPase geranylgeranylation in the mevalonate pathway. Our data underscore the importance of the negative regulator CD9 in lung inflammation, and suggest that statins exert anti-inflammatory effects by upregulating tetraspanin CD9 in macrophages.

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Three-dimensional culture of sebaceous gland cells revealing the role of prostaglandin E₂-induced activation of canonical Wnt signaling

Yoshida Go J., Saya Hideyuki

Biochemical and Biophysical Research Communications 438 ( 4 ) 640 - 646 2013.09

ISSN 0006-291X

Background: Prostaglandin E₂ (PGE₂) is a proinflammatory mediator and activates the canonical Wnt-β-catenin signaling pathway in hematopoietic stem cells. The SZ95 cell line was established from human sebaceous gland cells and is studied as a model system for these cells. Given that 2D culture of SZ95 cells does not recapitulate the organization of sebaceous glands in situ, we developed a 3D culture system for these cells and examined the effects of PGE₂ on cell morphology and function. Results: SZ95 cells maintained in 3D culture formed organoids that mimicked the organization of sebaceous glands in situ, including the establishment of a basement membrane. Organoids exposed to PGE₂ were larger and adopted a more complex organization compared with control organoids. PGE₂ activated the canonical Wnt signaling pathway as well as increased cell viability and proliferation, mitochondrial metabolism, and lipid synthesis in the organoids. Conclusions: Culture of SZ95 cells in 3D culture system recapitulates the structure and susceptibility to PGE₂ of sebaceous glands in situ and should prove useful for studies of the response of these glands to inflammation and other environmental stressors. Our results also implicate PGE₂-induced activation of canonical Wnt signaling pathway in regulation of the morphology,proliferation, and function of "semi-vivo" sebaceous glands.

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Energy management by enhanced glycolysis in G₁-phase in human colon cancer cells in vitro and in vivo

Bao Yan, Mukai Kuniaki, Hishiki Takako, Kubo Akiko, Ohmura Mitsuyo, Sugiura Yuki, Matsuura Tomomi, Nagahata Yoshiko, Hayakawa Noriyo, Yamamoto Takehiro, Fukuda Ryo, Saya Hideyuki, Suematsu Makoto, Minamishima Yoji Andrew

Molecular Cancer Research 11 ( 9 ) 973 - 985 2013.09

ISSN 1541-7786

Activation of aerobic glycolysis in cancer cells is well known as theWarburg effect, although its relation to cellcycle progression remains unknown. In this study, human colon cancer cells were labeled with a cell-cycle phasedependent fluorescent marker Fucci to distinguish cells in G1-phase and those in S⁺G2/M phases. Fucci-labeled cells served as splenic xenograft transplants in super-immunodeficient NOG mice and exhibited multiple metastases in the livers, frozen sections of which were analyzed by semiquantitative microscopic imaging mass spectrometry. Results showed that cells in G1-phase exhibited higher concentrations of ATP, NADH, and UDPN- acetylglucosamine than those in S andG2-Mphases, suggesting accelerated glycolysis inG1-phase cells in vivo. Quantitative determination of metabolites in cells synchronized in S, G2-M, and G1 phases suggested that efflux of lactate was elevated significantly in G1-phase. By contrast, ATP production in G2-Mwas highly dependent on mitochondrial respiration, whereas cells in S-phase mostly exhibited an intermediary energy metabolism between G1 and G2-Mphases. Isogenic cells carrying a p53-null mutation appeared more active in glycolysis throughout the cell cycle than wild-type cells. Thus, as the cell cycle progressed from G2-M toG1 phases, the dependency of energy production on glycolysis was increased while the mitochondrial energy production was reciprocally decreased. Implications: These results shed light on distinct features of the phase-specific phenotypes of metabolic systems in cancer cells.

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Acquired expression of NFATc1 downregulates e-cadherin and promotes cancer cell invasion

Oikawa Tsukasa, Nakamura Atsuko, Onishi Nobuyuki, Yamada Taketo, Matsuo Koichi, Saya Hideyuki

Cancer Research 73 ( 16 ) 5100 - 5109 2013.08

ISSN 0008-5472

NFATc1 is a transcription factor that regulates T-cell development, osteoclastogenesis, and macrophage function. Given that T cells, osteoclasts, and macrophages in the tumor microenvironment are thought to modulate tumor progression, tumor cells may acquire NFATc1 expression through fusion with these NFATc1- expressing normal cells. We here revealed that a small proportion of tumor cells in human carcinoma specimens expressed NFATc1. To investigate the consequences of NFATc1 acquisition by tumor cells, we established A549 and MCF7 cell lines expressing a constitutively active form of NFATc1 (NFATc1CA) in an inducible manner. The expression of NFATc1CA promoted cancer cell invasion in association with changes in cell morphology. Analysis of gene expression andRNAinterference experiments revealed that NFATc1CA suppressed E-cadherin expression by upregulating the transcriptional repressors Snail and Zeb1 in a manner independent of TGF-b signaling. Induced expression of NFATc1CA also downregulated E-cadherin expression and increased invasive activity in tumor xenografts in vivo. Our results thus suggest that the acquisition of NFATc1 expression contributes to tumor progression.

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CD44 variant 9 expression in primary early gastric cancer as a predictive marker for recurrence

Hirata K., Suzuki H., Imaeda H., Matsuzaki J., Tsugawa H., Nagano O., Asakura K., Saya H., Hibi T.

British Journal of Cancer 109 ( 2 ) 379 - 386 2013.07

ISSN 0007-0920

Background:Multiple early gastric cancers (EGCs) may develop in 6-14% of patients even after achieving curative endoscopic submucosal dissection (ESD); however, a useful biomarker for predicting recurrence is not available. The present study investigated whether the expression of CD44 variant 9 (CD44v9), a functional cancer stem cell marker, in the primary gastric cancer tissue represents an indicator of recurrence.Methods:Eighty-eight patients who underwent ESD for EGC from 2008 to 2010 were enrolled and monitored for recurrence for 3 years. The expression levels of CD44v9 in the tissue of initial EGCs were evaluated by immunohistochemistry, and the recurrence rate was compared between CD44v9-positive and CD44v9-negative groups. The mucin phenotype and expression of microRNA-21 (miR-21) and programmed cell death protein 4 (PDCD4) were also analysed.Results:The recurrence rate of EGC was significantly higher in the CD44v9-positive group than in the CD44v9-negative group (hazard ratio (HR), 21.8; 95% confidence interval (CI), 5.71-83.1). However, mucin phenotypes and the expression of miR-21 and PDCD4 did not predict recurrence after ESD. Meanwhile, grade of gastric atrophy was also identified as a significant marker of multiple recurrence (HR, 4.95; 95% CI, 1.30-18.8).Conclusion:CD44 variant 9 expression represents a potential predictive marker for recurrence in EGC.

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Twist2 functions as a tumor suppressor in murine osteosarcoma cells

Ishikawa Tomoki, Shimizu Takatsune, Ueki Arisa, Yamaguchi Sayaka I., Onishi Nobuyuki, Sugihara Eiji, Kuninaka Shinji, Miyamoto Takeshi, Morioka Hideo, Nakayama Robert, Kobayashi Eisuke, Toyama Yoshiaki, Mabuchi Yo, Matsuzaki Yumi, Yamaguchi Rui, Miyano Satoru, Saya Hideyuki

Cancer Science 104 ( 7 ) 880 - 888 2013.07

ISSN 1347-9032

The epithelial-mesenchymal transition (EMT) contributes to the malignant progression of cancer cells including acquisition of the ability to undergo metastasis. However, whereas EMT-related transcription factors (EMT-TF) are known to play an important role in the malignant progression of epithelial tumors, their role in mesenchymal tumors remains largely unknown. We show that expression of the gene for Twist2 is downregulated in human osteosarcoma and correlates inversely with tumorigenic potential in mouse osteosarcoma. Forced expression of Twist2 in highly tumorigenic murine osteosarcoma cells induced a slight inhibition of cell growth in vitro but markedly suppressed tumor formation in vivo. Conversely, knockdown of Twist2 in osteosarcoma cells with a low tumorigenic potential promoted tumor formation in vivo, suggesting that Twist2 functions as a tumor suppressor in osteosarcoma cells. Furthermore, Twist2 induced expression of fibulin-5, which has been reported as a tumor suppressor. Medium conditioned by mouse osteosarcoma cells overexpressing Twist2 inhibited expression of the MMP9 gene as well as invasion in mouse embryonic fibroblasts, and forced expression of Twist2 in osteosarcoma cells suppressed MMP9 gene expression in tumor tissue. Data from the present study suggest that Twist2 inhibits formation of a microenvironment conducive to tumor growth and thereby attenuates tumorigenesis in osteosarcoma.

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Targeting Aurora B to the Equatorial Cortex by MKlp2 Is Required for Cytokinesis

Kitagawa Mayumi, Fung Suet Yin Sarah, Onishi Nobuyuki, Saya Hideyuki, Lee Sang Hyun

PLoS One 8 ( 6 ) 2013.06

Although Aurora B is important in cleavage furrow ingression and completion during cytokinesis, the mechanism by which kinase activity is targeted to the cleavage furrow and the molecule(s) responsible for this process have remained elusive. Here, we demonstrate that an essential mitotic kinesin MKlp2 requires myosin-II for its localization to the equatorial cortex, and this event is required to recruit Aurora B to the equatorial cortex in mammalian cells. This recruitment event is also required to promote the highly focused accumulation of active RhoA at the equatorial cortex and stable ingression of the cleavage furrow in bipolar cytokinesis. Specifically, in drug-induced monopolar cytokinesis, targeting Aurora B to the cell cortex by MKlp2 is essential for cell polarization and furrow formation. Once the furrow has formed, MKlp2 further recruits Aurora B to the growing furrow. This process together with continuous Aurora B kinase activity at the growing furrow is essential for stable furrow propagation and completion. In contrast, a MKlp2 mutant defective in binding myosin-II does not recruit Aurora B to the cell cortex and does not promote furrow formation during monopolar cytokinesis. This mutant is also defective in maintaining the ingressing furrow during bipolar cytokinesis. Together, these findings reveal that targeting Aurora B to the cell cortex (or the equatorial cortex) by MKlp2 is essential for the maintenance of the ingressing furrow for successful cytokinesis.

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CAMP/PKA signalling reinforces the LATS-YAP pathway to fully suppress YAP in response to actin cytoskeletal changes

Kim Minchul, Kim Miju, Lee Seunghee, Kuninaka Shinji, Saya Hideyuki, Lee Ho, Lee Sookyung, Lim Dae Sik

EMBO Journal 32 ( 11 ) 1543 - 1555 2013.05

ISSN 0261-4189

Actin cytoskeletal damage induces inactivation of the oncoprotein YAP (Yes-associated protein). It is known that the serine/threonine kinase LATS (large tumour suppressor) inactivates YAP by phosphorylating its Ser127 and Ser381 residues. However, the events downstream of actin cytoskeletal changes that are involved in the regulation of the LATS-YAP pathway and the mechanism by which LATS differentially phosphorylates YAP on Ser127 and Ser381 in vivo have remained elusive. Here, we show that cyclic AMP (cAMP)-dependent protein kinase (PKA) phosphorylates LATS and thereby enhances its activity sufficiently to phosphorylate YAP on Ser381. We also found that PKA activity is involved in all contexts previously reported to trigger the LATS-YAP pathway, including actin cytoskeletal damage, G-protein-coupled receptor activation, and engagement of the Hippo pathway. Inhibition of PKA and overexpression of YAP cooperate to transform normal cells and amplify neural progenitor pools in developing chick embryos. We also implicate neurofibromin 2 as an AKAP (A-kinase-anchoring protein) scaffold protein that facilitates the function of the cAMP/PKA-LATS-YAP pathway. Our study thus incorporates PKA as novel component of the Hippo pathway.

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APC^CDH1 Targets MgcRacGAP for Destruction in the Late M Phase

Nishimura Koutarou, Oki Toshihiko, Kitaura Jiro, Kuninaka Shinji, Saya Hideyuki, Sakaue-Sawano Asako, Miyawaki Atsushi, Kitamura Toshio

PLoS One 8 ( 5 ) 2013.05

Background:Male germ cell RacGTPase activating protein (MgcRacGAP) is an important regulator of the Rho family GTPases - RhoA, Rac1, and Cdc42 - and is indispensable in cytokinesis and cell cycle progression. Inactivation of RhoA by phosphorylated MgcRacGAP is an essential step in cytokinesis. MgcRacGAP is also involved in G1-S transition and nuclear transport of signal transducer and activator of transcription 3/5 (STAT3/5). Expression of MgcRacGAP is strictly controlled in a cell cycle-dependent manner. However, the underlying mechanisms have not been elucidated.Methodology/Principal Findings:Using MgcRacGAP deletion mutants and the fusion proteins of full-length or partial fragments of MgcRacGAP to mVenus fluorescent protein, we demonstrated that MgcRacGAP is degraded by the ubiquitin-proteasome pathway in the late M to G1 phase via APC^CDH1. We also identified the critical region for destruction located in the C-terminus of MgcRacGAP, AA537-570, which is necessary and sufficient for CDH1-mediated MgcRacGAP destruction. In addition, we identified a PEST domain-like structure with charged residues in MgcRacGAP and implicate it in effective ubiquitination of MgcRacGAP.Conclusions/Significance:Our findings not only reveal a novel mechanism for controlling the expression level of MgcRacGAP but also identify a new target of APC^CDH1. Moreover our results identify a C-terminal region AA537-570 of MgcRacGAP as its degron.

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The Arf/p53 protein module, which induces apoptosis, down-regulates histone H2AX to allow normal cells to survive in the presence of anti-cancer drugs

Atsumi Yuko, Inase Aki, Osawa Tomoyuki, Sugihara Eiji, Sakasai Ryo, Fujimori Hiroaki, Teraoka Hirobumi, Saya Hideyuki, Kanno Masamoto, Tashiro Fumio, Nakagama Hitoshi, Masutani Mitsuko, Yoshioka Ken Ichi

Journal of Biological Chemistry 288 ( 19 ) 13269 - 13277 2013.05

ISSN 0021-9258

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Correction

Muto Jun, Imai Takao, Ogawa Daisuke, Nishimoto Yoshinori, Okada Yohei, Mabuchi Yo, Kawase Takeshi, Iwanami Akio, Mischel Paul S., Saya Hideyuki, Yoshida Kazunari, Matsuzaki Yumi, Okano Hideyuki

PLoS One 8 ( 4 ) 2013.04

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IGF1 receptor signaling regulates adaptive radioprotection in glioma stem cells

Osuka Satoru, Sampetrean Oltea, Shimizu Takatsune, Saga Isako, Onishi Nobuyuki, Sugihara Eiji, Okubo Jun, Fujita Satoshi, Takano Shingo, Matsumura Akira, Saya Hideyuki

Stem Cells 31 ( 4 ) 627 - 640 2013.04

ISSN 1066-5099

Cancer stem cells (CSCs) play an important role in disease recurrence after radiation treatment as a result of intrinsic properties such as high DNA repair capability and antioxidative capacity. It is unclear, however, how CSCs further adapt to escape the toxicity of the repeated irradiation regimens used in clinical practice. Here, we have exposed a population of murine glioma stem cells (GSCs) to fractionated radiation in order to investigate the associated adaptive changes, with the ultimate goal of identifying a targetable factor that regulates acquired radioresistance. We have shown that fractionated radiation induces an increase in IGF1 secretion and a gradual upregulation of the IGF type 1 receptor (IGF1R) in GSCs. Interestingly, IGF1R upregulation exerts a dual radioprotective effect. In the resting state, continuous IGF1 stimulation ultimately induces downregulation of Akt/ extracellular-signal-regulated kinases (ERK) and FoxO3a activation, which results in slower proliferation and enhanced self-renewal. In contrast, after acute radiation, the abundance of IGF1R and increased secretion of IGF1 promote a rapid shift from a latent state toward activation of Akt survival signaling, protecting GSCs from radiation toxicity. Treatment of tumors formed by the radioresistant GSCs with an IGF1R inhibitor resulted in a marked increase in radiosensitivity, suggesting that blockade of IGF1R signaling is an effective strategy to reverse radioresistance. Together, our results show that GSCs evade the damage of repeated radiation not only through innate properties but also through gradual inducement of resistance pathways and identify the dynamic regulation of GSCs by IGF1R signaling as a novel mechanism of adaptive radioprotection.

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XCT inhibition depletes CD44v-expressing tumor cells that are resistant to EGFR-targeted therapy in head and neck squamous cell carcinoma

Yoshikawa Momoko, Tsuchihashi Kenji, Ishimoto Takatsugu, Yae Toshifumi, Motohara Takeshi, Sugihara Eiji, Onishi Nobuyuki, Masuko Takashi, Yoshizawa Kunio, Kawashiri Shuichi, Mukai Makio, Asoda Seiji, Kawana Hiromasa, Nakagawa Taneaki, Saya Hideyuki, Nagano Osamu

Cancer Research 73 ( 6 ) 1855 - 1866 2013.03

ISSN 0008-5472

The targeting of antioxidant systems that allow stem-like cancer cells to avoid the adverse consequences of oxidative stress might be expected to improve the efficacy of cancer treatment. Here, we show that head and neck squamous cell carcinoma (HNSCC) cells that express variant isoforms of CD44 (CD44v) rely on the activity of the cystine transporter subunit xCT for control of their redox status. xCT inhibition selectively induces apoptosis in CD44v-expressing tumor cells without affecting CD44v-negative differentiated cells in the same tumor. In contrast to CD44v-expressing undifferentiated cells, CD44v-negative differentiated cells manifest EGF receptor (EGFR) activation and rely on EGFR activity for their survival. Combined treatment with inhibitors of xCT-dependent cystine transport and of EGFR resulted in a synergistic reduction of EGFR-expressing HNSCC tumor growth. Thus, xCT-targeted therapy may deplete CD44v-expressing undifferentiated HNSCC cells and concurrently sensitize the remaining differentiating cells to available treatments including EGFR-targeted therapy.

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Complexity of cancer stem cells

Sugihara Eiji, Saya Hideyuki

International Journal of Cancer 132 ( 6 ) 1249 - 1259 2013.03

ISSN 0020-7136

Heterogeneity of tumor tissue has been accounted for in recent years by a hierarchy-based model in which cancer stem cells (CSCs) have the ability both to self-renew and to give rise to differentiated tumor cells and are responsible for the overall organization of a tumor. Research into CSCs has progressed rapidly and concomitantly with recent advances in the biology of normal tissue stem cells, resulting in the identification of CSCs in a wide range of human tumors. Studies of mouse models of human cancer have provided further insight into the characteristics of CSCs as well as a basis for the development of novel therapies targeted to these cells. However, recent studies have revealed complexities, such as plasticity of stem cell properties and clonal diversity of CSCs, in certain tumor types that have led to revision of the original CSC model. In this review, we summarize the history of the discovery and characterization of CSCs, as well as address recent advances that have revealed the complexity of these cells and their therapeutic implications.

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Arf and p53 act as guardians of a quiescent cellular state by protecting against immortalization of cells with stable genomes

Osawa Tomoyuki, Atsumi Yuko, Sugihara Eiji, Saya Hideyuki, Kanno Masamoto, Tashiro Fumio, Masutani Mitsuko, Yoshioka Ken ichi

Biochemical and Biophysical Research Communications 432 ( 1 ) 34 - 39 2013.03

ISSN 0006-291X

Normal cells undergo a growth-arrested status that is produced by p53-dependent down-regulation of histone H2AX. Immortality is developed after abrogation of the H2AX-diminished state, which is associated with genomic instability (often with tetraploidy) and the induction of mutations in either the Arf or p53 gene. However, the role of Arf in control of H2AX expression and genome stability is still unclear. Here, we show that both Arf and p53 are required for the down-regulation of H2AX and formation of the growth-arrested state. Wild-type (WT) mouse embryonic fibroblasts (MEFs) subjected to tetraploidization with DNA lesions did not undergo mitotic catastrophe-associated cell death and stayed in a growth-arrested state, until immortality was attained with mutations in the Arf/. p53 module and recovery of H2AX expression. Whereas tetraploidization was essential for immortalization of WT MEFs, this event was not required for immortalization of MEFs containing mutations in Arf/. p53 and these cells still underwent mitotic catastrophe-associated cell death. Thus, WT MEFs are protected from immortalization with genome stability, which is abrogated with tetraploidization and mutation of either Arf or p53.

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Gene expression profile of a newly established choriocarcinoma cell line, iC³-1, compared to existing choriocarcinoma cell lines and normal placenta

Kobayashi Y., Banno K., Shimizu T., Ueki A., Tsuji K., Masuda K., Kisu I., Nomura H., Tominaga E., Nagano O., Saya H., Aoki D.

Placenta 34 ( 2 ) 110 - 118 2013.02

ISSN 0143-4004

Gestational choriocarcinoma is a malignant trophoblastic tumor that usually occurs in the uterus after pregnancy. The tumor is curable with advanced chemotherapy, but the molecular mechanism of choriocarcinoma tumorigenesis remains unclear. This is partly because the low incidence makes it difficult to obtain clinical samples for investigation and because an appropriate choriocarcinoma cell model to study the tumorigenesis has not been developed. We have established a new choriocarcinoma cell line, induced choriocarcinoma cell-1 (iC³-1), that possesses unique characteristics compared to other choriocarcinoma cell lines, including production of tumors that consist of the two types of cells commonly found in choriocarcinoma and mimicking of the clinical pathology. Existing trophoblast cell lines utilized in previous choriocarcinoma studies have had significantly dissimilar gene expression profiles. Therefore, it is important to choose an appropriate cell line for a particular study based on the characteristics of the cell line. In this study, to clarify the genetic characteristics of iC³-1 and to explore the tumorigenesis mechanism, we examined the gene profile of iC³-1 compared to those of existing cell lines and normal placental tissue. Bioinformatics analysis showed that several characteristic genes, IGF1R, CHFR, MUC3A, TAF7, PARK7, CDC123 and PSMD8, were significantly upregulated in iC ³-1 compared to BeWo and JEG3 cells. Interestingly, HAS2, CD44 and S100P were significantly upregulated in iC³-1 compared to parental HTR8/SVneo cells and normal third trimester placenta. Choriocarcinoma samples also showed immunoreactivity to HAS2, CD44 and S100. In summary, the gene expression profile of iC³-1 suggests that studies using this cell line can make an important contribution to improved understanding of choriocarcinoma tumorigenesis.

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Establishment of HRAS^G12V Transgenic Medaka as a Stable Tumor Model for In Vivo Screening of Anticancer Drugs

Matsuzaki Yuriko, Hosokai Haru, Mizuguchi Yukiyo, Fukamachi Shoji, Shimizu Atsushi, Saya Hideyuki

PLoS One 8 ( 1 ) 2013.01

Most targeted anticancer drugs have been identified by screening at the molecular or cellular level in vitro. However, many compounds selected by such costly and time-consuming screening do not prove effective against tumors in vivo. The development of anticancer drugs would thus be facilitated by the availability of an in vivo screening system based on a multicellular organism. We have now established a transgenic line of the freshwater fish medaka in which melanophores (melanocytes) proliferate in a manner dependent on heat shock-induced signaling by a human RAS oncoprotein. The human HRAS^G12V oncogene was expressed under the control of a melanophore-specific gene promoter in order to allow visualization of tumor growth in live fish maintained in a water tank. The expression of HRAS^G12V was induced as a result of Cre-mediated recombination by exposure of the fish to a temperature of 37°C for 30 min, given that the Cre gene was placed under the control of a medaka heat shock promoter. One of the stable transgenic lines developed abnormal pigment cell proliferation in the eyes and epidermis with 100% penetrance by 6 months postfertilization. Sorafenib, an inhibitor of RAS signaling, was administered to the transgenic fish and was found both to reduce the extent of melanophore proliferation and to improve survival. The transgenic medaka established here thus represents a promising in vivo system with which to screen potential anticancer drugs that target RAS signaling, and this system can readily be adapted for the screening of agents that target other oncogenes.

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The APC/C activator Cdh1 regulates the G2/M transition during differentiation of placental trophoblast stem cells

Naoe Hideaki, Chiyoda Tatsuyuki, Ishizawa Jo, Masuda Kenta, Saya Hideyuki, Kuninaka Shinji

Biochemical and Biophysical Research Communications 430 ( 2 ) 757 - 762 2013.01

ISSN 0006-291X

Differentiation of placental trophoblast stem (TS) cells to trophoblast giant (TG) cells is accompanied by transition from a mitotic cell cycle to an endocycle. Here, we report that Cdh1, a regulator of the anaphase-promoting complex/cyclosome (APC/C), negatively regulates mitotic entry upon the mitotic/endocycle transition. TS cells derived from homozygous Cdh1 gene-trapped (Cdh1^GT/GT) murine embryos accumulated mitotic cyclins and precociously entered mitosis after induction of TS cell differentiation, indicating that Cdh1 is required for the switch from mitosis to the endocycle. Furthermore, the Cdh1^GT/GT TS cells and placenta showed aberrant expression of placental differentiation markers. These data highlight an important role of Cdh1 in the G2/M transition during placental differentiation.

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Identification of novel small compounds that restore E-cadherin expression and inhibit tumor cell motility and invasiveness

Hirano Tamaki, Satow Reiko, Kato Asami, Tamura Mana, Murayama Yumi, Saya Hideyuki, Kojima Hirotatsu, Nagano Tetsuo, Okabe Takayoshi, Fukami Kiyoko

Biochemical Pharmacology 86 ( 10 ) 1419 - 1429 2013

ISSN 0006-2952

Tumor dissemination and invasive behavior are associated with a majority of cancer-related mortality cases. Loss of E-cadherin, which is caused by several tumor-promoting factors, is associated with metastasis and poor prognosis in many neoplasms. In this study, we aimed to identify small molecule compounds that restore the expression of E-cadherin, because these molecules are most likely to suppress tumor malignancy by restoring E-cadherin function and/or by inhibiting signals that suppress E-cadherin expression. Here, we developed a fluorescence screen system based on E-cadherin expression. A pilot drug library screen revealed that methotrexate (MTX) strongly induces E-cadherin expression in a colorectal cancer cell line, SW620. From the screen for 9600 compounds, we identified 9 hit compounds, which restored the expression of E-cadherin in SW620 and/or a melanoma cell line, SK-MEL-28. We confirmed that MTX and the other identified compounds transcriptionally promote E-cadherin expression. Among these, 2 compounds suppressed migration/invasion capacity in colorectal cancer cells and 3 in melanoma cells. A compound reduced SW620 migration and invasion with subtle effects on cell viability in SW620, SK-MEL-28, and a non-tumor cell line, HaCaT, with decrease in AKT and ERK1/2 protein levels. One of the other compounds reduced SK-MEL-28 cell migration and invasion and affected the viability only of SW620 and SK-MEL-28 cells but not HaCaT cells. These results suggest that these compounds would be attractive lead molecules as anti-metastasis agents.

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In vivo pH monitoring using boron doped diamond microelectrode and silver needles

Fierro Stéphane, Seishima Ryo, Nagano Osamu, Saya Hideyuki, Einaga Yasuaki

Scientific Reports 3 2013

This study presents the in vivo electrochemical monitoring of pH using boron doped diamond (BDD) microelectrode and silver needles for potential application in medical diagnosis. Accurate calibration curve for pH determination were obtained through in vitro electrochemical measurements. The increase induced in stomach pH by treatment with pantoprazole was used to demonstrate that it is possible to monitor the pH in vivo using the simple and noninvasive system proposed herein. Using the results of the in vivo and in vitro experiments, a quantitative analysis of the increase in stomach pH is also presented. It is proposed that the catheter-free pH monitoring system presented in this study could be potentially employed in any biological environment.

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Lysyl oxidase is induced by cell density-mediated cell cycle suppression via RB-E2F1-HIF-1α axis

Goto Takaaki M., Arima Yoshimi, Nagano Osamu, Saya Hideyuki

Cell Structure and Function 38 ( 1 ) 9 - 14 2013

ISSN 0386-7196

Remodeling of the matrix surrounding tumor cells plays a crucial role in the development and maintenance of cancer. Lysyl oxidase (LOX), a matrix remodeling factor, is induced by HIF-1α under hypoxic conditions and associated with tumor growth and metastasis. Here, we report that high cell density induces HIF-1α expression under normoxic condition, resulting in the promotion of LOX expression. This phenomenon was observed in the retinoblastoma tumor suppressor (RB)-proficient breast cancer cells but not in RB-deficient cells. In RB-proficient cancer cells, the cell cycle regulator E2F1 was down-regulated and cell cycle progression was inhibited at high density culture condition. Knockdown of E2F1 stabilized HIF-1α and promoted LOX expression, while knockdown of both E2F1 and HIF-1α prevented the up-regulation of LOX. These findings suggest that elevated cell density enhances cell cycle arrest and matrix remodeling via RB-E2F1-HIF-1α axis.

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A microfluidic invasion assay for glioma-initiating cells in three-dimensional culture

Fujioka Shingo, Oltea Sampetrean, Saya Hideyuki, Sudo Ryo

1075 - 1077 2013

This paper presents a microfluidic three-dimensional (3D) platform to investigate behaviors of glioma-initiating cells (GIC) cocultured with human umbilical vein endothelial cells (HUVEC). In this platform, these cells were cultured in microenvironments similar to in vivo. Especially, our experiments have been performed with GIC established by gene transfection [1], which is different from conventional tumor cell lines. Our results demonstrate that biochemical factors secreted by HUVEC attracts glioma invasion.

Reactive oxygen species-induced autophagic degradation of helicobacter pylori CagA is specifically suppressed in cancer stem-like cells

Tsugawa Hitoshi, Suzuki Hidekazu, Saya Hideyuki, Hatakeyama Masanori, Hirayama Toshiya, Hirata Kenro, Nagano Osamu, Matsuzaki Juntaro, Hibi Toshifumi

Cell Host and Microbe 12 ( 6 ) 764 - 777 2012.12

ISSN 1931-3128

Sustained expression of CagA, the type IV secretion effector of Helicobacter pylori, is closely associated with the development of gastric cancer. However, we observed that after translocation, CagA is degraded by autophagy and therefore short lived. Autophagy and CagA degradation are induced by the H. pylori vacuolating cytotoxin, VacA, which acted via decreasing intracellular glutathione (GSH) levels, causing reactive oxygen species (ROS) accumulation and Akt activation. Investigating this further, we found that CagA specifically accumulated in gastric cells expressing CD44, a cell-surface marker associated with cancer stem cells. The autophagic pathway in CD44-positive gastric cancer stem-like cells is suppressed because of their resistance to ROS, which is supported by increased intracellular GSH levels. These findings provide a molecular link between H. pylori and gastric carcinogenesis through the specific accumulation of CagA in gastric cancer stem-like cells.

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Tumor necrosis factor-α (TNF-α) stimulates the epithelial-mesenchymal transition regulator Snail in cholangiocarcinoma

Techasen Anchalee, Namwat Nisana, Loilome Watcharin, Bungkanjana Pornpan, Khuntikeo Narong, Puapairoj Anucha, Jearanaikoon Patcharee, Saya Hideyuki, Yongvanit Puangrat

Medical Oncology 29 ( 5 ) 3083 - 3091 2012.12

ISSN 1357-0560

Epithelial-mesenchymal transition (EMT) is a series of events during which epithelial cells lose many of their epithelial characteristics and take on properties that are typical of mesenchymal cells that lack cell-cell adhesion properties. EMT may be activated by various types of growth factors or inflammatory cytokines. In many types of epithelial cancers, the EMT-derived tumor cells are susceptible to metastasis. During tumor progression, epithelial cells acquire a gene expression pattern closely resembling that of mesenchymal cells. This study aimed to investigate the expression of the EMT-associated transcription factor Snail and an adhesion molecule E-cadherin in cholangiocarcinoma (CCA) tissues. The effect of TNF-α on EMT activation in CCA cells was also demonstrated. The qRT-PCR analysis revealed that Snail expression significantly increased in CCA (P = 0.01) and was correlated with tumor metastasis (P = 0.02). The expression of Snail was inversely associated with E-cadherin (P = 0.004). The stimulation of TNF-α enhances migration behavior and showed significantly induced expression of Snail in CCA cell lines, whereas expression of E-cadherin and CK-19 (the epithelial marker) was reduced. Immunofluorescence analysis revealed that TNF-α-treated CCA cell lines increased nuclear translocation of Snail, whereas E-cadherin was dramatically decreased. Our findings suggest that the changes in the expression of Snail or E-cadherin might regulate EMT development in CCA resulting in promoting tumor progression. Overexpression of Snail could be used as a prognostic marker for monitoring the treatment efficiency of CCA patients.

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Up-Regulation of Imp3 Confers In Vivo Tumorigenicity on Murine Osteosarcoma Cells

Ueki Arisa, Shimizu Takatsune, Masuda Kenta, Yamaguchi Sayaka I., Ishikawa Tomoki, Sugihara Eiji, Onishi Nobuyuki, Kuninaka Shinji, Miyoshi Keita, Muto Akihiro, Toyama Yoshiaki, Banno Kouji, Aoki Daisuke, Saya Hideyuki

PLoS One 7 ( 11 ) 2012.11

Osteosarcoma is a high-grade malignant bone tumor that manifests ingravescent clinical behavior. The intrinsic events that confer malignant properties on osteosarcoma cells have remained unclear, however. We previously established two lines of mouse osteosarcoma cells: AX cells, which are able to form tumors in syngeneic mice, and AXT cells, which were derived from such tumors and acquired an increased tumorigenic capacity during tumor development. We have now identified Igf2 mRNA-binding protein3 (Imp3) as a key molecule responsible for this increased tumorigenicity of AXT cells in vivo. Imp3 is consistently up-regulated in tumors formed by AX cells, and its expression in these cells was found to confer malignant properties such as anchorage-independent growth, loss of contact inhibition, and escape from anoikis in vitro. The expression level of Imp3 also appeared directly related to tumorigenic ability in vivo which is the critical determination for tumor-initiating cells. The effect of Imp3 on tumorigenicity of osteosarcoma cells did not appear to be mediated through Igf2-dependent mechanism. Our results implicate Imp3 as a key regulator of stem-like tumorigenic characteristics in osteosarcoma cells and as a potential therapeutic target for this malignancy.

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Reciprocal control of G ₁-phase progression is required for Th-POK/Runx3-mediated CD4/8 thymocyte cell fate decision

Sato Takehito, Chiba Tomoki, Ohno Shin Ichiro, Sato Chiharu, Sugoh Tatsuya, Miyashita Keiko, Akatsuka Hisako, Hozumi Katsuto, Okada Yoshinori, Iida Yumi, Akatsuka Akira, Agata Yasutoshi, Chiba Marin, Kohu Kazuyoshi, Satake Masanobu, Tanabe Hideyuki, Saya Hideyuki, Habu Sonoko

Journal of Immunology 189 ( 9 ) 4426 - 4436 2012.11

ISSN 0022-1767

After receiving a TCR-mediated differentiation signal, CD4 and CD8 double-positive thymocytes diverge into CD4 or CD8 single-positive T cells, for which Th-POK and Runx3 have been identified as pivotal transcription factors, respectively. The cross-antagonistic regulation of Th-POK and Runx3 seems to be essential for CD4/8 thymocyte lineage commitment. However, the process for determining which pivotal factor acts dominantly has not been established. To explore the determining process, we used an in vitro culture system in which CD4 or CD8 single-positive cells are selectively induced from CD4/8 double-positive cells. Surprisingly, we found that control of G ₁ cell cycle phase progression is critical for the determination. In the CD4 pathway, sustained TCR signal, as well as Th-POK, induces G ₁-phase extension and represses CD8 expression in a G ₁ extension-dependent manner. In the CD8 pathway, after receiving a transient TCR signal, the IL-7R signal, as well as Runx3, antagonizes TCR signal-mediated G ₁ extension and CD8 repression. Importantly, forced G ₁ extension cancels the functions of Runx3 to repress Th-POK and CD4 and to reactivate CD8. In contrast, it is suggested that forced G ₁ progression inhibits Th-POK function to repress CD8. Collectively, Th-POK and Runx3 are reciprocally involved in the control of G ₁-phase progression, on which they exert their functions dependently. These findings may provide novel insight into how CD4/CD8 cell lineages are determined by Th-POK and Runx3.

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Pyruvate kinase M2

Tamada Mayumi, Suematsu Makoto, Saya Hideyuki

Clinical Cancer Research 18 ( 20 ) 5554 - 5561 2012.10

ISSN 1078-0432

The M2 splice isoform of pyruvate kinase (PKM2), an enzyme that catalyzes the later step of glycolysis, is a key regulator of aerobic glycolysis (known as the Warburg effect) in cancer cells. Expression and low enzymatic activity of PKM2 confer on cancer cells the glycolytic phenotype, which promotes rapid energy production and flow of glycolytic intermediates into collateral pathways to synthesize nucleic acids, amino acids, and lipids without the accumulation of reactive oxygen species.PKM2 enzymatic activity has also been shown to be negatively regulated by the interaction with CD44 adhesion molecule, which is a cell surface marker for cancer stem cells. In addition to the glycolytic functions, nonglycolytic functions of PKM2 in cancer cells are of particular interest. PKM2 is induced translocation into the nucleus, where it activates transcription of various genes by interacting with and phosphorylating specific nuclear proteins, endowing cancer cells with a survival and growth advantage. Therefore, inhibitors and activators of PKM2 are well underway to evaluate their anticancer effects and suitability for use as novel therapeutic strategies.

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Novel strategies for cancer stem cells

Saya Hideyuki

International Journal of Biological Markers 27 ( 4 ) 2012.10

ISSN 0393-6155

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[Therapeutic strategy for breast cancer stem cells].

Yoshida G. J., Saya Hideyuki

Nihon rinsho. Japanese journal of clinical medicine 70 Suppl 7 243 - 248 2012.09

ISSN 0047-1852

CD44s regulates the TGF-β-mediated mesenchymal phenotype and is associated with poor prognosis in patients with hepatocellular carcinoma

Mima Kosuke, Okabe Hirohisa, Ishimoto Takatsugu, Hayashi Hiromitsu, Nakagawa Shigeki, Kuroki Hideyuki, Watanabe Masayuki, Beppu Toru, Tamada Mayumi, Nagano Osamu, Saya Hideyuki, Baba Hideo

Cancer Research 72 ( 13 ) 3414 - 3423 2012.07

ISSN 0008-5472

The prognosis for individuals diagnosed with hepatocellular carcinoma (HCC) remains poor because of the high frequency of invasive tumor growth, intrahepatic spread, and extrahepatic metastasis. Here, we investigated the role of the standard isoform of CD44 (CD44s), a major adhesion molecule of the extracellular matrix and a cancer stem cell marker, in the TGF-β-mediated mesenchymal phenotype of HCC. We found that CD44s was the dominant form of CD44 mRNA expressed in HCC cells. Overexpression of CD44s promoted tumor invasiveness and increased the expression of vimentin, a mesenchymal marker, in HCC cells. Loss of CD44s abrogated these changes. Also in the setting of CD44s overexpression, treatment with TGF-β1 induced the mesenchymal phenotype of HCC cells, which was characterized by low E-cadherin and high vimentin expression. Loss of CD44s inhibited TGF-β-mediated vimentin expression, mesenchymal spindle-like morphology, and tumor invasiveness. Clinically, overexpression of CD44s was associated with low expression of E-cadherin, high expression of vimentin, a high percentage of phospho-Smad2-positive nuclei, and poor prognosis in HCC patients, including reduced disease-free and overall survival. Together, our findings suggest that CD44s plays a critical role in the TGF-β-mediated mesenchymal phenotype and therefore represents a potential therapeutic target for HCC.

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Ink4a and Arf are crucial factors in the determination of the cell of origin and the therapeutic sensitivity of Myc-induced mouse lymphoid tumor

Sugihara E., Shimizu T., Kojima K., Onishi N., Kai K., Ishizawa J., Nagata K., Hashimoto N., Honda H., Kanno M., Miwa M., Okada S., Andreeff M., Saya H.

Oncogene 31 ( 23 ) 2849 - 2861 2012.06

ISSN 0950-9232

The cell of origin of tumors and the factors determining the cell of origin remain unclear. In this study, a mouse model of precursor B acute lymphoblastic leukemia/lymphoma (pre-B ALL/LBL) was established by retroviral transduction of Myc genes (N-Myc or c-Myc) into mouse bone marrow cells. Hematopoietic stem cells (HSCs) exhibited the highest susceptibility to N-Myc-induced pre-B ALL/LBL versus lymphoid progenitors, myeloid progenitors and committed progenitor B cells. N-Myc was able to induce pre-B ALL/LBL directly from progenitor B cells in the absence of Ink4a and Arf. Arf was expressed higher in progenitor B cells than Ink4a. In addition, N-Myc induced pre-B ALL/LBL from Arf/progenitor B cells suggesting that Arf has a predominant role in determining the cell of origin of pre-B ALL/LBL. Tumor cells derived from Ink4a/Arf/progenitor B cells exhibited a higher rate of proliferation and were more chemoresistant than those derived from wild-type HSCs. Furthermore, the Mdm2 inhibitor Nutlin-3 restored p53 and induced massive apoptosis in mouse pre-B ALL/LBL cells derived from Ink4a/Arf/cells and human B-ALL cell lines lacking Ink4a and Arf expression, suggesting that Mdm2 inhibition may be a novel therapeutic approach to the treatment of Ink4a/Arf/B-ALL/LBL, such as is frequently found in Ph ALL and relapsed ALL. Collectively, these findings indicate that Ink4a and Arf are critical determining factors of the cell of origin and the therapeutic sensitivity of Myc-induced lymphoid tumors.

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Loss of p16 expression is associated with the stem cell characteristics of surface markers and therapeutic resistance in estrogen receptor-negative breast cancer

Arima Yoshimi, Hayashi Naoki, Hayashi Hidemi, Sasaki Mikako, Kai Kazuharu, Sugihara Eiji, Abe Eriko, Yoshida Atsushi, Mikami Shuji, Nakamura Seigo, Saya Hideyuki

International Journal of Cancer 130 ( 11 ) 2568 - 2579 2012.06

ISSN 0020-7136

Triple-negative breast cancer [TNBC, which is negative for the estrogen receptor (ER), progesterone receptor, and human epidermal growth factor receptor 2] is a high-risk form of the disease without a specific therapy. DNA microarray and immunohistochemical analyses have shown that most TNBCs fall within the basal-like histological subset of breast cancers, which frequently exhibit inactivation of the retinoblastoma tumor suppressor (Rb) and upregulation of the cyclin-dependent kinase inhibitor p16 ^INK4a (p16). However, downregulation of p16 expression has been observed in some basal-like breast cancer cell lines, suggesting that such cells can be divided into two groups according to Rb and p16 status. We now show that cells that are CD44 ⁺ and CD24 ^-, a phenotype associated with stem-like breast cancer cells, are more abundant in ER ^-/p16 ^- breast cancer cell lines than in ER ^-/p16 ⁺ lines. It was also found that p16 expression was downregulated in mammospheres from an ER-negative breast cancer cell line. Depletion of p16 by RNA interference in ER-negative breast cancer cells increased the percentage of CD44 ⁺/CD24 ^- cells and increased the expression of mRNA of the ES-like genes Nanog, Oct4, and Sox2 through an Rb-independent pathway. Furthermore, such depletion of p16 reduced chemosensitivity. The loss of p16 expression may thus reduce the response of ER-negative breast cancer cells to chemotherapy by conferring cancer stem cell-like properties. Consistent with this conclusion, immunohistochemical analysis of the clinical samples suggests that low p16 expression in TNBC is associated with resistance to preoperative chemotherapy.

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LATS1/WARTS phosphorylates MYPT1 to counteract PLK1 and regulate mammalian mitotic progression

Chiyoda Tatsuyuki, Sugiyama Naoyuki, Shimizu Takatsune, Naoe Hideaki, Kobayashi Yusuke, Ishizawa Jo, Arima Yoshimi, Tsuda Hiroshi, Ito Masaaki, Kaibuchi Kozo, Aoki Daisuke, Ishihama Yasushi, Saya Hideyuki, Kuninaka Shinji

Journal of Cell Biology 197 ( 5 ) 625 - 641 2012.05

ISSN 0021-9525

In the mitotic exit network of budding yeast, Dbf2 kinase phosphorylates and regulates Cdc14 phosphatase. In contrast, no phosphatase substrates of LATS1/WARTS kinase, the mammalian equivalent of Dbf2, has been reported. To address this discrepancy, we performed phosphoproteomic screening using LATS1 kinase. Screening identified MYPT1 (myosin phosphatase- targeting subunit 1) as a new substrate for LATS1. LATS1 directly and preferentially phosphorylated serine 445 (S445) of MYPT1. An MYPT1 mutant (S445A) failed to dephosphorylate Thr 210 of PLK1 (pololike kinase 1), thereby activating PLK1. This suggests that LATS1 promotes MYPT1 to antagonize PLK1 activity. Consistent with this, LATS1-depleted HeLa cells or fibroblasts from LATS1 knockout mice showed increased PLK1 activity. We also found deoxyribonucleic acid (DNA) damage-induced LATS1 activation caused PLK1 suppression via the phosphorylation of MYPT1 S445. Furthermore, LATS1 knockdown cells showed reduced G2 checkpoint arrest after DNA damage. These results indicate that LATS1 phosphorylates a phosphatase as does the yeast Dbf2 and demonstrate a novel role of LATS1 in controlling PLK1 at the G2 DNA damage checkpoint.

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Tks5-dependent formation of circumferential podosomes/invadopodia mediates cell-cell fusion

Oikawa Tsukasa, Oyama Masaaki, Kozuka-Hata Hiroko, Uehara Shunsuke, Udagawa Nobuyuki, Saya Hideyuki, Matsuo Koichi

Journal of Cell Biology 197 ( 4 ) 553 - 568 2012.05

ISSN 0021-9525

Osteoclasts fuse to form multinucleated cells during osteoclastogenesis. This process is mediated by dynamic rearrangement of the plasma membrane and cytoskeleton, and it requires numerous factors, many of which have been identified. The underlying mechanism remains obscure, however. In this paper, we show that Tks5, a master regulator of invadopodia in cancer cells, is crucial for osteoclast fusion downstream of phosphoinositide 3-kinase and Src. Expression of Tks5 was induced during osteoclastogenesis, and prevention of this induction impaired both the formation of circumferential podosomes and osteoclast fusion without affecting cell differentiation. Tyrosine phosphorylation of Tks5 was attenuated in Src ^-/- osteoclasts, likely accounting for defects in podosome organization and multinucleation in these cells. Circumferential invadopodia formation in B16F0 melanoma cells was also accompanied by Tks5 phosphorylation. Co-culture of B16F0 cells with osteoclasts in an inflammatory milieu promoted the formation of melanoma-osteoclast hybrid cells. Our results thus reveal an unexpected link between circumferential podosome/invadopodium formation and cell-cell fusion in and beyond osteoclasts.

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Modulation of glucose metabolism by CD44 contributes to antioxidant status and drug resistance in cancer cells

Tamada Mayumi, Nagano Osamu, Tateyama Seiji, Ohmura Mitsuyo, Yae Toshifumi, Ishimoto Takatsugu, Sugihara Eiji, Onishi Nobuyuki, Yamamoto Takehiro, Yanagawa Hiroshi, Suematsu Makoto, Saya Hideyuki

Cancer Research 72 ( 6 ) 1438 - 1448 2012.03

ISSN 0008-5472

An increased glycolytic flux accompanied by activation of the pentose phosphate pathway (PPP) is implicated in chemoresistance of cancer cells. In this study, we found that CD44, a cell surface marker for cancer stem cells, interacts with pyruvate kinase M2 (PKM2) and thereby enhances the glycolytic phenotype of cancer cells that are either deficient in p53 or exposed to hypoxia. CD44 ablation by RNA interference increased metabolic flux to mitochondrial respiration and concomitantly inhibited entry into glycolysis and the PPP. Such metabolic changes induced by CD44 ablation resulted in marked depletion of cellular reduced glutathione (GSH) and increased the intracellular level of reactive oxygen species in glycolytic cancer cells. Furthermore, CD44 ablation enhanced the effect of chemotherapeutic drugs in p53-deficient or hypoxic cancer cells. Taken together, our findings suggest that metabolic modulation by CD44 is a potential therapeutic target for glycolytic cancer cells that manifest drug resistance.

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RNA-binding protein musashi1 modulates glioma cell growth through the post-transcriptional regulation of notch and PI ₃ kinase/Akt signaling pathways

Muto Jun, Imai Takao, Ogawa Daisuke, Nishimoto Yoshinori, Okada Yohei, Mabuchi Yo, Kawase Takeshi, Iwanami Akio, Mischel Paul S., Saya Hideyuki, Yoshida Kazunari, Matsuzaki Yumi, Okano Hideyuki

PLoS One 7 ( 3 ) 2012.03

Musashi1 (MSI1) is an RNA-binding protein that plays critical roles in nervous-system development and stem-cell self-renewal. Here, we examined its role in the progression of glioma. Short hairpin RNA (shRNA)-based MSI1-knock down (KD) in glioblastoma and medulloblastoma cells resulted in a significantly lower number of self renewing colony on day 30 (a 65% reduction), compared with non-silencing shRNA-treated control cells, indicative of an inhibitory effect of MSI1-KD on tumor cell growth and survival. Immunocytochemical staining of the MSI1-KD glioblastoma cells indicated that they ectopically expressed metaphase markers. In addition, a 2.2-fold increase in the number of MSI1-KD cells in the G2/M phase was observed. Thus, MSI1-KD caused the prolongation of mitosis and reduced the cell survival, although the expression of activated Caspase-3 was unaltered. We further showed that MSI1-KD glioblastoma cells xenografted into the brains of NOD/SCID mice formed tumors that were 96.6% smaller, as measured by a bioluminescence imaging system (BLI), than non-KD cells, and the host survival was longer (49.3±6.1 days vs. 33.6±3.6 days; P>0.01). These findings and other cell biological analyses suggested that the reduction of MSI1 in glioma cells prolonged the cell cycle by inducing the accumulation of Cyclin B1. Furthermore, MSI1-KD reduced the activities of the Notch and PI ₃ kinase-Akt signaling pathways, through the up-regulation of Numb and PTEN, respectively. Exposure of glioma cells to chemical inhibitors of these pathways reduced the number of spheres and living cells, as did MSI1-KD. These results suggest that MSI1 increases the growth and/or survival of certain types of glioma cells by promoting the activation of both Notch and PI ₃ kinase/Akt signaling.

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Induction of ZEB proteins by inactivation of RB protein is key determinant of mesenchymal phenotype of breast cancer

Arima Yoshimi, Hayashi Hidemi, Sasaki Mikako, Hosonaga Mari, Goto Takaaki M., Chiyoda Tatsuyuki, Kuninaka Shinji, Shibata Tatsuhiro, Ohata Hirokazu, Nakagama Hitoshi, Taya Yoichi, Saya Hideyuki

Journal of Biological Chemistry 287 ( 11 ) 7896 - 7906 2012.03

ISSN 0021-9258

Wepreviously showed that depletion of the retinoblastoma protein (RB) induces down-regulation of the adhesion molecule E-cadherin and thereby triggers the epithelial-mesenchymal transition. To further characterize the effect of RB inactivation on the phenotype of cancer cells, we have now examined RB expression in human breast cancer cell lines and clinical specimens. We found that RB-inactive cells exhibit a mesenchymal-like morphology and are highly invasive. We also found that ZEB proteins, transcriptional repressors of the E-cadherin gene, are markedly up-regulated in these cells in a manner sensitive to the miR-200 family of microRNAs. Moreover, depletion of ZEB in RB-inactive cells suppressed cell invasiveness and proliferation and induced epithelial marker expression. These results implicate ZEB in induction of the epithelial-mesenchymal transition, as well as in maintenance of the mesenchymal phenotype in RB-inactive cells. We also developed a screening program for inhibitors of ZEB1 expression and thereby identified several cyclin-dependent kinase inhibitors that blocked both ZEB1 expression and RB phosphorylation. Together, our findings suggest that RB inactivation contributes to tumor progression not only through loss of cell cycle control but also through up-regulation of ZEB expression and induction of an invasive phenotype.

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Fibroblast growth factor-2 is an important factor that maintains cellular immaturity and contributes to aggressiveness of osteosarcoma

Shimizu Takatsune, Ishikawa Tomoki, Iwai Sayaka, Ueki Arisa, Sugihara Eiji, Onishi Nobuyuki, Kuninaka Shinji, Miyamoto Takeshi, Toyama Yoshiaki, Ijiri Hiroshi, Mori Hajime, Matsuzaki Yumi, Yaguchi Tomonori, Nishio Hiroshi, Kawakami Yutaka, Ikeda Yasuo, Saya Hideyuki

Molecular Cancer Research 10 ( 3 ) 454 - 468 2012.03

ISSN 1541-7786

Osteosarcoma is the most frequent, nonhematopoietic, primary malignant tumor of bone. Histopathologically, osteosarcoma is characterized by complex mixtures of different cell types with bone formation. The role of environmental factors in the formation of such a complicated tissue structure as osteosarcoma remains to be elucidated. Here, a newly established murine osteosarcoma model was used to clarify the roles of environmental factors such as fibroblast growth factor-2 (Fgf2) or leukemia-inhibitory factor (Lif) in the maintenance of osteosarcoma cells in an immature state. These factors were highly expressed in tumor environmental stromal cells, rather than in osteosarcoma cells, and they potently suppressed osteogenic differentiation of osteosarcoma cells in vitro and in vivo. Further investigation revealed that the hyperactivation of extracellular signal-regulated kinase (Erk)1/2 induced by these factors affected in the process of osteosarcoma differentiation. In addition, Fgf2 enhanced both proliferation and migratory activity of osteosarcoma cells and modulated the sensitivity of cells to an anticancer drug. The results of the present study suggest that the histology of osteosarcoma tumors which consist of immature tumor cells and pathologic bone formations could be generated dependent on the distribution of such environmental factors. The combined blockade of the signaling pathways of several growth factors, including Fgf2, might be useful in controlling the aggressiveness of osteosarcoma.

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Expression profiles of carcinosarcoma of the uterine corpus-are these similar to carcinoma or sarcoma?

Chiyoda Tatsuyuki, Tsuda Hiroshi, Tanaka Hideo, Kataoka Fumio, Nomura Hiroyuki, Nishimura Sadako, Takano Masashi, Susumu Nobuyuki, Saya Hideyuki, Aoki Daisuke

Genes Chromosomes and Cancer 51 ( 3 ) 229 - 239 2012.03

ISSN 1045-2257

Uterine carcinosarcoma (CS) is usually classified as uterine endometrial carcinoma (EC). However, CS is more aggressive even compared with high grade EC. CS is also reported to undergo epithelial to mesenchymal transition (EMT). In this study, we compared the gene expression profiles of CS, EC, and uterine sarcoma (US) and evaluated the role of EMT and chromosomal aberrations in CS tumor formation. Frozen tissues of 46 patients (14 CS, 24 EC, and 8 US) were included. The similarity was examined by Gene Set Enrichment Analysis (GSEA), Fisher's exact test, and clustering using "intrinsic gene set". We examined the expression of 39 EMT-related genes and evaluated TGF-beta signaling by phospho-SMAD2/3 (p-SMAD2/3) staining. Chromosomal regions differing between CS and EC were identified by chromosomal GSEA and comparative genomic hybridization (CGH) microarrays. Three statistical methods confirmed that CS resembled US rather than EC. Acquired markers of EMT were upregulated and attenuated markers of EMT were downregulated in CS. Immunohistochemistry showed that carcinomatous region of CS have higher expression of p-SMAD2/3 than EC (P = 0.008). Chromosomal GSEA showed that genes located at 19q13 had higher expression in CS. Furthermore, CGH microarray indicated that the TGFB1 locus at 19q13.1 was amplified in 4 of 7 samples. Based on the expression profile, CS resembles US rather than EC. TGF-beta signaling is activated in CS and chromosomal gains at 19q13, which includes the TGFB1 locus, suggest that this may contribute to high expression of TGF-beta and thereby EMT phenotype of CS.

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Anti-tumor effect against human cancer xenografts by a fully human monoclonal antibody to a variant 8-epitope of CD44R1 expressed on cancer stem cells

Masuko Kazue, Okazaki Shogo, Satoh Mayumi, Tanaka Goh, Ikeda Tatsuya, Torii Ryota, Ueda Eri, Nakano Takashi, Danbayashi Masaaki, Tsuruoka Tomoyo, Ohno Yoshiya, Yagi Hideki, Yabe Noritsugu, Yoshida Hideaki, Tahara Tomoyuki, Kataoka Shiro, Oshino Taichi, Shindo Takayuki, Niwa Shin ichiro, Ishimoto Takatsugu, Baba Hideo, Hashimoto Yoshiyuki, Saya Hideyuki, Masuko Takashi

PLoS One 7 ( 1 ) 2012.01

Background: CD44 is a major cellular receptor for hyaluronic acids. The stem structure of CD44 encoded by ten normal exons can be enlarged by ten variant exons (v1-v10) by alternative splicing. We have succeeded in preparing MV5 fully human IgM and its class-switched GV5 IgG monoclonal antibody (mAb) recognizing the extracellular domain of a CD44R1 isoform that contains the inserted region coded by variant (v8, v9 and v10) exons and is expressed on the surface of various human epithelial cancer cells. Methods and Principal Findings: We demonstrated the growth inhibition of human cancer xenografts by a GV5 IgG mAb reshaped from an MV5 IgM. The epitope recognized by MV5 and GV5 was identified to a v8-coding region by the analysis of mAb binding to various recombinant CD44 proteins by enzyme-linked immunosorbent assay. GV5 showed preferential reactivity against various malignant human cells versus normal human cells assessed by flow cytometry and immunohistological analysis. When ME180 human uterine cervix carcinoma cells were subcutaneously inoculated to athymic mice with GV5, significant inhibition of tumor formation was observed. Furthermore, intraperitoneal injections of GV5markedly inhibited the growth of visible established tumors from HSC-3 human larynx carcinoma cells that had been subcutaneously transplanted one week before the first treatment with GV5. From in vitro experiments, antibody-dependent cellular cytotoxicity and internalization of CD44R1 seemed to be possible mechanisms for in vivo anti-tumor activity by GV5. Conclusions: CD44R1 is an excellent molecular target for mAb therapy of cancer, possibly superior to molecules targeted by existing therapeutic mAb, such as Trastuzumab and Cetuximab recognizing human epidermal growth factor receptor family.

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DNA damage signaling triggers degradation of histone methyltransferases through APC/C ^Cdh1 in senescent cells

Takahashi Akiko, Imai Yoshinori, Yamakoshi Kimi, Kuninaka Shinji, Ohtani Naoko, Yoshimoto Shin, Hori Satoshi, Tachibana Makoto, Anderton Emma, Takeuchi Takashi, Shinkai Yoichi, Peters Gordon, Saya Hideyuki, Hara Eiji

Molecular Cell 45 ( 1 ) 123 - 131 2012.01

ISSN 1097-2765

Both the DNA damage response (DDR) and epigenetic mechanisms play key roles in the implementation of senescent phenotypes, but very little is known about how these two mechanisms are integrated to establish senescence-associated gene expression. Here we show that, in senescent cells, the DDR induces proteasomal degradation of G9a and GLP, major histone H3K9 mono- and dimethyltransferases, through Cdc14B- and p21 ^Waf1/Cip1-dependent activation of APC/C ^Cdh1 ubiquitin ligase, thereby causing a global decrease in H3K9 dimethylation, an epigenetic mark for euchromatic gene silencing. Interestingly, induction of IL-6 and IL-8, major players of the senescence-associated secretory phenotype (SASP), correlated with a decline of H3K9 dimethylation around the respective gene promoters and knockdown of Cdh1 abolished IL-6/IL-8 expression in senescent cells, suggesting that the APC/C ^Cdh1-G9a/GLP axis plays crucial roles in aspects of senescent phenotype. These findings establish a role for APC/C ^Cdh1 and reveal how the DDR integrates with epigenetic processes to induce senescence-associated gene expression.

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In vivo assessment of cancerous tumors using boron doped diamond microelectrode

Fierro Stéphane, Yoshikawa Momoko, Nagano Osamu, Yoshimi Kenji, Saya Hideyuki, Einaga Yasuaki

Scientific Reports 2 2012

The in vitro and in vivo electrochemical detection of the reduced form of glutathione (L-γ-glutamyl-L-cysteinyl-glycine, GSH) using boron doped diamond (BDD) microelectrode for potential application in the assessment of cancerous tumors is presented. Accurate calibration curve for the determination of GSH could be obtained by the in vitro electrochemical measurements. Additionally, it was shown that it was possible to separate the detection of GSH from the oxidized form of glutathione (GSSG) using chronoamperometry measurements. In vivo GSH detection measurements have been performed in human cancer cells inoculated in immunodeficient mice. These measurements have shown that the difference of GSH level between cancerous and normal tissues can be detected. Moreover, GSH detection measurements carried out before and after X-ray irradiation have proved that it is possible to assess in vivo the decrease in GSH concentration in the tumor after a specific treatment.

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Alternative splicing of CD44 mRNA by ESRP1 enhances lung colonization of metastatic cancer cell

Yae Toshifumi, Tsuchihashi Kenji, Ishimoto Takatsugu, Motohara Takeshi, Yoshikawa Momoko, Yoshida Go J., Wada Takeyuki, Masuko Takashi, Mogushi Kaoru, Tanaka Hiroshi, Osawa Tsuyoshi, Kanki Yasuharu, Minami Takashi, Aburatani Hiroyuki, Ohmura Mitsuyo, Kubo Akiko, Suematsu Makoto, Takahashi Kazuhisa, Saya Hideyuki, Nagano Osamu

Nature Communications 3 2012

In cancer metastasis, various environmental stressors attack the disseminating cells. The successful colonization of cancer cells in secondary sites therefore requires the ability of the cells to avoid the consequences of such exposure to the stressors. Here we show that orthotopic transplantation of a CD44 variant isoform-expressing (CD44v⁺) subpopulation of 4T1 breast cancer cells, but not that of a CD44v^- subpopulation, in mice results in efficient lung metastasis accompanied by expansion of stem-like cancer cells. Such metastasis is dependent on the activity of the cystine transporter xCT, and the stability of this protein is controlled by CD44v. We find that epithelial splicing regulatory protein 1 regulates the expression of CD44v, and knockdown of epithelial splicing regulatory protein 1 in CD44v ⁺ cells results in an isoform switch from CD44v to CD44 standard (CD44s), leading to reduced cell surface expression of xCT and suppression of lung colonization. The epithelial splicing regulatory protein 1-CD44v-xCT axis is thus a potential therapeutic target for the prevention of metastasis.

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Molecular biology of glioma

Marumoto Tomotoshi, Saya Hideyuki

2 - 11 2012

Glioblastoma (GBM) is the most aggressive form of glioma. Despite ceaseless efforts by researchers and physicians to find new therapeutic strategies, there have been no significant advances in the treatment of GBMs for several decades and most patients with GBM die within one and half years of diagnosis. Undoubtedly, one reason for this is the insufficient understanding of the initiation and progression of GBMs at the molecular level. However, recent information regarding the genetic and epigenetic alterations and the microRNAs that are aberrantly activated or inactivated in GBMs has helped elucidate the formation of GBM in more detail. Here, we describe recent advances in the understanding of the biology of GBMs.

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Erratum

Tomita M., Toyota M., Ishikawa C., Nakazato T., Okudaira T., Matsuda T., Uchihara J. N., Taira N., Ohshiro K., Senba M., Tanaka Y., Ohshima K., Saya H., Tokino T., Mori N.

International Journal of Cancer 129 ( 11 ) 2763 2011.12

ISSN 0020-7136

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Transient depletion of p53 followed by transduction of c-Myc and K-Ras converts ovarian stem-like cells into tumor-initiating cells

Motohara Takeshi, Masuko Sachiko, Ishimoto Takatsugu, Yae Toshifumi, Onishi Nobuyuki, Muraguchi Teruyuki, Hirao Atsushi, Matsuzaki Yumi, Tashiro Hironori, Katabuchi Hidetaka, Saya Hideyuki, Nagano Osamu

Carcinogenesis 32 ( 11 ) 1597 - 1606 2011.11

ISSN 0143-3334

Although the existence of tumor-initiating cells (T-ICs) in several types of human cancer has been documented, the contribution of somatic stem cells to the development of T-ICs has remained unclear. Here, we show that normal mouse ovary contains epithelial cell adhesion molecule (EpCAM)-expressing stem-like cells that possess the ability to differentiate into cytokeratin 8 (CK8)-expressing epithelial progeny cells. Furthermore, RNA interference-mediated transient depletion of the tumor suppressor p53 followed by retrovirus-mediated transfer of c-Myc and K-Ras oncogenes in EpCAM-expressing ovarian stem-like cells resulted in the generation of ovarian T-ICs. The established ovarian T-ICs gave rise to hierarchically organized lethal tumors in vivo and were able to undergo peritoneal metastasis. Finally, subsequent RNA interference-mediated knockdown of p53 in tumor cells triggered the expansion of EpCAM-expressing stem-like tumor cells and induced further tumor growth. These data reveal a role for p53 in the development and expansion of ovarian stem-like tumor cells and subsequent malignant progression.

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Invasion precedes tumor mass formation in a malignant brain tumor model of genetically modified neural stem cells

Sampetrean Oltea, Saga Isako, Nakanishi Masaya, Sugihara Eiji, Fukaya Raita, Onishi Nobuyuki, Osuka Satoru, Akahata Masaki, Kai Kazuharu, Sugimoto Hachiro, Hirao Atsushi, Saya Hideyuki

Neoplasia 13 ( 9 ) 784 - 791 2011.09

ISSN 1522-8002

Invasiveness, cellular atypia, and proliferation are hallmarks of malignant gliomas. To effectively target each of these characteristics, it is important to understand their sequence during tumorigenesis. However, because most gliomas are diagnosed at an advanced stage, the chronology of gliomagenesis milestones is not well understood. The aim of the present study was to determine the onset of these characteristics during tumor development. Brain tumor-initiating cells (BTICs) were established by overexpressing H-Ras ^V12 in normal neural stem/progenitor cells isolated from the subventricular zone of adult mice harboring a homozygous deletion of the Ink4a/Arf locus. High-grade malignant brain tumors were then created by orthotopic implantation of 10 ⁵ BTICs into the forebrain of 6-week-old wild-type mice. Micewere killed every week for 5 weeks, and tumors were assessed for cellular atypia, proliferation, hemorrhage, necrosis, and invasion. All mice developed highly invasive, hypervascular glioblastoma-like tumors. A 100% penetrance rate and a 4-week median survival were achieved. Tumor cell migration along fiber tracts started within days after implantation and was followed by perivascular infiltration of tumor cells with marked recruitment of reactive host cells. Next, cellular atypia became prominent. Finally, mass proliferation and necrosis were observed in the last stage of the disease. Video monitoring of BTICs in live brain slices confirmed the early onset of migration, as well as the main cell migration patterns. Our results showed that perivascular and intraparenchymal tumor cell migration precede tumor mass formation in the adult brain, suggesting the need for an early and sustained anti-invasion therapy.

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Establishment of a choriocarcinoma model from immortalized normal extravillous trophoblast cells transduced with HRASV12

Kobayashi Yusuke, Shimizu Takatsune, Naoe Hideaki, Ueki Arisa, Ishizawa Joe, Chiyoda Tatsuyuki, Onishi Nobuyuki, Sugihara Eiji, Nagano Osamu, Banno Kouji, Kuninaka Shinji, Aoki Daisuke, Saya Hideyuki

American Journal of Pathology 179 ( 3 ) 1471 - 1482 2011.09

ISSN 0002-9440

Gestational choriocarcinoma is a malignant trophoblastic tumor. The development of novel molecular-targeted therapies is needed to reduce the toxicity of current multiagent chemotherapy and to treat successfully the chemoresistant cases. The molecular mechanisms underlying choriocarcinoma tumorigenesis remain uncharacterized, however, and appropriate choriocarcinoma animal models have not yet been developed. In this study, we established a choriocarcinoma model by inoculating mice with induced-choriocarcinoma cell1 (iC ³-1) cells, generated from HTR8/SVneo human trophoblastic cells retrovirally transduced with activated H-RAS (HRASV12). The iC ³-1 cells exhibited constitutive activation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways and developed into lethal tumors in all inoculated mice. Histopathological analysis revealed that the tumors consisted of two distinct types of cells, reminiscent of syncytiotrophoblasts and cytotrophoblasts, as seen in the human choriocarcinoma. The tumors expressed HLA-G and cytokeratin (trophoblast markers) and hCG (a choriocarcinoma marker). Comparative analysis of gene expression profiles between iC ³-1 cells and parental HTR8/SVneo cells revealed that iC ³-1 cells expressed matrix metalloproteinases, epithelialmesenchymal transition-related genes, and SOX3 at higher levels than parental trophoblastic cells. Administration of SOX3-specific short-hairpin RNA decreased SOX3 expression and attenuated the tumorigenic activity of iC ³-1 cells, suggesting that SOX3 overexpression might be critically involved in the pathogenesis of choriocarcinoma. Our murine model represents a potent new tool for studying the pathogenesis and treatment of choriocarcinoma.

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PRKAR1A is overexpressed and represents a possible therapeutic target in human cholangiocarcinoma

Loilome Watcharin, Juntana Sirinun, Namwat Nisana, Bhudhisawasdi Vajarabhongsa, Puapairoj Anucha, Sripa Banchob, Miwa Masanao, Saya Hideyuki, Riggins Gregory J., Yongvanit Puangrat

International Journal of Cancer 129 ( 1 ) 34 - 44 2011.07

ISSN 0020-7136

The protein kinase A regulatory subunit 1 alpha (PRKAR1A/PKAI) pathway is overexpressed in varieties of tumors and cancer cell lines including cholangiocarcinoma (CCA), although its role in CCA growth modulation is unclear. In our study, we evaluated the effect of PRKAR1A/PKAI targeting on CCA cell proliferation. Real-time PCR demonstrated an increased mRNA expression of PRKAR1A/PKAI, whereas protein kinase A regulatory subunit 2 beta (PRKAR2B/PKAII) was downregulated in human CCA tissues and CCA cell lines. Immunohistochemistry of human CCA tissues revealed increased PRKAR1A with decreased PRKAR2B protein expression. Moreover, CCA cell lines showed abundantly expressed PRKAR1A, while lacking PRKAR2B expression. Silencing PRKAR1A expression induced growth inhibition and apoptosis of CCA cells, with an associated decrease in mitogen-activated protein kinases, PI3K/Akt, JAK/STAT and Wnt/β-catenin pathway signaling. The inhibition of PKA using a PKA inhibitor and cAMP analogs also led to a significant cell growth inhibition. In conclusion, our study reports the overexpression as well as molecular mechanisms by which PRKAR1A/PKA regulates human CCA cell growth. Importantly, abrogation of gene expression caused significant CCA cell growth inhibition, oncogenic signaling and coupled apoptosis induction, suggesting PRKAR1A's potential as a drug target for CCA therapy.

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Functional analysis of HOXD9 in human gliomas and glioma cancer stem cells

Tabuse Masanao, Ohta Shigeki, Ohashi Yohei, Fukaya Raita, Misawa Aya, Yoshida Kazunari, Kawase Takeshi, Saya Hideyuki, Thirant Cécile, Chneiweiss Hérve, Matsuzaki Yumi, Okano Hideyuki, Kawakami Yutaka, Toda Masahiro

Molecular Cancer 10 2011.05

Background: HOX genes encode a family of homeodomain-containing transcription factors involved in the determination of cell fate and identity during embryonic development. They also behave as oncogenes in some malignancies.Results: In this study, we found high expression of the HOXD9 gene transcript in glioma cell lines and human glioma tissues by quantitative real-time PCR. Using immunohistochemistry, we observed HOXD9 protein expression in human brain tumor tissues, including astrocytomas and glioblastomas. To investigate the role of HOXD9 in gliomas, we silenced its expression in the glioma cell line U87 using HOXD9-specific siRNA, and observed decreased cell proliferation, cell cycle arrest, and induction of apoptosis. It was suggested that HOXD9 contributes to both cell proliferation and/or cell survival. The HOXD9 gene was highly expressed in a side population (SP) of SK-MG-1 cells that was previously identified as an enriched-cell fraction of glioma cancer stem-like cells. HOXD9 siRNA treatment of SK-MG-1 SP cells resulted in reduced cell proliferation. Finally, we cultured human glioma cancer stem cells (GCSCs) from patient specimens found with high expression of HOXD9 in GCSCs compared with normal astrocyte cells and neural stem/progenitor cells (NSPCs).Conclusions: Our results suggest that HOXD9 may be a novel marker of GCSCs and cell proliferation and/or survival factor in gliomas and glioma cancer stem-like cells, and a potential therapeutic target.

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The cell cycle regulator Cdh1 controls the pool sizes of hematopoietic stem cells and mature lineage progenitors by protecting from genotoxic stress

Ishizawa Jo, Kuninaka Shinji, Sugihara Eiji, Naoe Hideaki, Kobayashi Yusuke, Chiyoda Tatsuyuki, Ueki Arisa, Araki Kimi, Yamamura Ken Ichi, Matsuzaki Yumi, Nakajima Hideaki, Ikeda Yasuo, Okamoto Shinichiro, Saya Hideyuki

Cancer Science 102 ( 5 ) 967 - 974 2011.05

ISSN 1347-9032

Various key cell cycle components, especially G0/G1 regulators, have effects not only on cell proliferation but also on cell differentiation. Cdh1, one of the co-activators that maintain anaphase-promoting complex/cyclosome activity, plays a crucial role in the mitotic phase, but has recently been identified as a G0/G1 regulator, suggesting that the role of Cdh1 in cell differentiation. Here, we generated Cdh1 conditional gene-trap mice to examine Cdh1 functions in adult tissues by overcoming the embryonic lethality of Cdh1 homozygous gene-trap mice. We focused on the hematopoietic system and found that Cdh1-deficient mice exhibited a general decrease in mature lineage progenitor cells and a significant increase in short-term hematopoietic stem cells. This phenomenon became conspicuous by irradiation shortly after Cdh1 downregulation, suggesting that Cdh1 regulates the pool sizes of the hematopoietic stem cells and mature lineage progenitor cells by protecting cells from genotoxic stress. We also found that the irradiation-induced G2/M checkpoint was defective in Cdh1-deficient BM cells, causing the loss of stem/progenitor cells. This is the first report revealing Cdh1 function in adult hematopoiesis and showing a role of Cdh1 in a G2/M checkpoint regulation in vivo.

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Oncogenicity of L-type amino-acid transporter 1 (LAT1) revealed by targeted gene disruption in chicken DT40 cells

Ohkawa Mayumi, Ohno Yoshiya, Masuko Kazue, Takeuchi Akiko, Suda Kentaro, Kubo Akihiro, Kawahara Rieko, Okazaki Shogo, Tanaka Toshiyuki, Saya Hideyuki, Seki Masayuki, Enomoto Takemi, Yagi Hideki, Hashimoto Yoshiyuki, Masuko Takashi

Biochemical and Biophysical Research Communications 406 ( 4 ) 649 - 655 2011.03

ISSN 0006-291X

L-type amino-acid transporter 1 (LAT1) is the first identified light chain of CD98 molecule, disulfide-linked to a heavy chain of CD98. Following cDNA cloning of chicken full-length LAT1, we have constructed targeting vectors for the disruption of chicken LAT1 gene from genomic DNA of chicken LAT1 consisting of 5.4kb. We established five homozygous LAT1-disrupted (LAT1^-/-) cell clones, derived from a heterozygous LAT1^+/- clone of DT40 chicken B cell line. Reactivity of anti-chicken CD98hc monoclonal antibody (mAb) with LAT1^-/- DT40 cells was markedly decreased compared with that of wild-type DT40 cells. All LAT1^-/- cells were deficient in L-type amino-acid transporting activity, although alternative-splice variant but not full-length mRNA of LAT1 was detected in these cells. LAT1^-/- DT40 clones showed outstandingly slow growth in liquid culture and decreased colony-formation capacity in soft agar compared with wild-type DT40 cells. Cell-cycle analyses indicated that LAT1^-/- DT40 clones have prolonged cell-cycle phases compared with wild-type or LAT1^+/- DT40 cells. Knockdown of human LAT1 by small interfering RNAs resulted in marked in vitro cell-growth inhibition of human cancer cells, and in vivo tumor growth of HeLa cells in athymic mice was significantly inhibited by anti-human LAT1 mAb. All these results indicate essential roles of LAT1 in the cell proliferation and occurrence of malignant phenotypes and that LAT1 is a promising candidate as a molecular target of human cancer therapy.

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CD44 Variant Regulates Redox Status in Cancer Cells by Stabilizing the xCT Subunit of System xc- and Thereby Promotes Tumor Growth

Ishimoto Takatsugu, Nagano Osamu, Yae Toshifumi, Tamada Mayumi, Motohara Takeshi, Oshima Hiroko, Oshima Masanobu, Ikeda Tatsuya, Asaba Rika, Yagi Hideki, Masuko Takashi, Shimizu Takatsune, Ishikawa Tomoki, Kai Kazuharu, Takahashi Eri, Imamura Yu, Baba Yoshifumi, Ohmura Mitsuyo, Suematsu Makoto, Baba Hideo, Saya Hideyuki

Cancer Cell 19 ( 3 ) 387 - 400 2011.03

ISSN 1535-6108

CD44 is an adhesion molecule expressed in cancer stem-like cells. Here, we show that a CD44 variant (CD44v) interacts with xCT, a glutamate-cystine transporter, and controls the intracellular level of reduced glutathione (GSH). Human gastrointestinal cancer cells with a high level of CD44 expression showed an enhanced capacity for GSH synthesis and defense against reactive oxygen species (ROS). Ablation of CD44 induced loss of xCT from the cell surface and suppressed tumor growth in a transgenic mouse model of gastric cancer. It also induced activation of p38 ^MAPK, a downstream target of ROS, and expression of the gene for the cell cycle inhibitor p21 ^CIP1/WAF1. These findings establish a function for CD44v in regulation of ROS defense and tumor growth.

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[Encounter of cancer cells with bone. The significance of cancer stem cells and epithelial-mesenchymal transition in tumor invasion and metastasis].

Yoshida G. J., Saya Hideyuki

Clinical calcium 21 ( 3 ) 411 - 417 2011.03

ISSN 0917-5857

Cancer stem cells (CSCs) , which are subset of tumor cells resistant to radiation and chemotherapy, are associated with malignant characteristics of tumor and possess both self-renewal ability and pluripotency for tumor formation. In the process of generating non-CSCs from CSCs, gene mutations and epigenetic changes are induced in those cells, resulting in composition of tumor tissue with heterogeneous cell population. CSCs have been recognized as the source of metastatic foci. Epithelial-mesenchymal transition (EMT) is a change in cellular phenotype characterized by the loss of cell-to-cell adhesions and the gain of migratory behaviors,which has been shown to be a critical factor for initiating cancer invasion and metastasis. However, some recent studies suggest that EMT is not essential requirement for tumor invasion and metastasis. Herein, we discuss the biological significance of CSCs and EMT in tumor invasion and metastasis.

NKX2.2 suppresses self-renewal of glioma-initiating cells

Muraguchi Teruyuki, Tanaka Shingo, Yamada Daisuke, Tamase Akira, Nakada Mitsutoshi, Nakamura Hideo, Hoshii Takayuki, Ooshio Takako, Tadokoro Yuko, Naka Kazuhito, Ino Yasushi, Todo Tomoki, Kuratsu Jun Ichi, Saya Hideyuki, Hamada Jun Ichiro, Hirao Atsushi

Cancer Research 71 ( 3 ) 1135 - 1145 2011.02

ISSN 0008-5472

Glioblastoma (GBM) is the most aggressive and destructive form of brain cancer. Animal models that can unravel the mechanisms underlying its progression are needed to develop rational and effective molecular therapeutic approaches. In this study, we report the development of mouse models for spontaneous gliomas representing distinct progressive stages of disease that are governed by defined genetic alterations. Neural stem/ progenitor cell (NPC)-specific constitutive Ras activation in vivo plus p53 deficiency led to development of primarily anaplastic astrocytoma (grade III), whereas combined loss of p53 plus p16^Ink4a/p19^Arf led to development of GBM (grade IV) at 100% penetrance within 6 weeks. These glioma models showed enhanced stem cell properties (stemness) accompanied by malignant progression. Notably, we determined that, in our models and in human specimens, downregulation of the homeodomain transcription factor NKX2.2, which is essential for oligodendroglial differentiation, was correlated with increased tumor malignancy. NKX2.2 overexpression by GBM-derived glioma-initiating cells (GIC) induced oligodendroglial differentiation and suppressed self-renewal capacity. By contrast, Nkx2.2 downregulation in mouse NPCs accelerated GBM formation. Importantly, the inhibitory effects of NXK2.2 on GIC self-renewal were conserved in human cells. Thus, our mouse models offer pathobiologically significant advantages to investigate the nature of brain tumors, with improved opportunities to develop novel mechanism-based therapeutic approaches.

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Mad2 inhibits the mitotic kinesin MKlp2

Lee Sang Hyun, McCormick Frank, Saya Hideyuki

Journal of Cell Biology 191 ( 6 ) 1069 - 1077 2010.12

ISSN 0021-9525

We identified the mitotic kinesin-like protein 2 (MKlp2), a kinesin required for chromosome passenger complex (CPC)-mediated cytokinesis, as a target of the mitotic checkpoint protein Mad2. MKlp2 possesses a consensus Mad2-binding motif required for Mad2 binding. Mad2 prevents MKlp2 from loading onto the mitotic spindle, a prerequisite step for its function as a mitotic kinesin. Furthermore, Mad2 inhibits the ability of MKlp2 to relocate the CPC from centromeres, an essential step to promote cytokinesis. An MKlp2 mutant that is refractory to Mad2-mediated inhibition prematurely translocates to the mitotic spindle and mislocalizes the CPC component Aurora B from the midbody of dividing cells. This correlates with an increased incidence of cytokinesis failure. Together, these findings reveal that MKlp2 is a novel mitotic target of Mad2 necessary for proper mitotic progression and cytokinesis.

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Identification of CD44 as a cell surface marker for Müller glia precursor cells

Shinoe Toru, Kuribayashi Hiroshi, Saya Hideyuki, Seiki Motoharu, Aburatani Hiroyuki, Watanabe Sumiko

Journal of Neurochemistry 115 ( 6 ) 1633 - 1642 2010.12

ISSN 0022-3042

In the retina, both neurons and glia differentiate from a common progenitor population. CD44 cell surface antigen is a hyaluronic acid receptor expressed on mature Müller glial cells. We found that in the developing mouse retina, expression of CD44 was transiently observed at or around birth in a subpopulation of c-kit-positive retinal progenitor cells. During in vitro culture, purified CD44/c-kit-positive retinal progenitor cells exclusively differentiated into Müller glial cells and not into neurons, suggesting that CD44 marks a subpopulation of retinal progenitor cells that are fated to become glia. Over-expression of CD44 inhibited the extension of processes by Müller glial cells and neurons. Notch signaling is known to be involved in the specification of retinal progenitors into a glial fate. Activation of Notch signaling increased the number of CD44-positive cells, and treatment with the Notch signal inhibitor, DAPT, at early, but not later, stages of retinal development abolished both CD44-positive cells and Müller glial cells. Together, CD44 was identified as an early cell surface marker of the Müller glia lineage, and Notch signalling was involved in commitment of retinal progenitor cells to CD44 positive Müller glial precursor cells.

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C-MYC overexpression with loss of Ink4a/Arf transforms bone marrow stromal cells into osteosarcoma accompanied by loss of adipogenesis

Shimizu T., Ishikawa T., Sugihara E., Kuninaka S., Miyamoto T., Mabuchi Y., Matsuzaki Y., Tsunoda T., Miya F., Morioka H., Nakayama R., Kobayashi E., Toyama Y., Kawai A., Ichikawa H., Hasegawa T., Okada S., Ito T., Ikeda Y., Suda T., Saya H.

Oncogene 29 ( 42 ) 5687 - 5699 2010.10

ISSN 0950-9232

The development of cancer is due to the growth and proliferation of transformed normal cells. Recent evidence suggests that the nature of oncogenic stress and the state of the cell of origin critically affect both tumorigenic activity and tumor histological type. However, this mechanistic relationship in mesenchymal tumors is currently largely unexplored. To clarify these issues, we established a mouse osteosarcoma (OS) model through overexpression of c-MYC in bone marrow stromal cells (BMSCs) derived from Ink4a/Arf (/) mice. Single-cell cloning revealed that c-MYC-expressing BMSCs are composed of two distinctly different clones: highly tumorigenic cells, similar to bipotent-committed osteochondral progenitor cells, and low-tumorigenic tripotent cells, similar to mesenchymal stem cells (MSCs). It is noteworthy that both bipotent and tripotent cells were capable of generating histologically similar, lethal OS, suggesting that both committed progenitor cells and MSCs can become OS cells of origin. Shifting mesenchymal differentiation by depleting PPARγ in tripotent MSC-like cells and overexpressing PPARγ in bipotent cells affected cell proliferation and tumorigenic activity. Our findings indicate that differentiation potential has a key role in OS tumorigenic activity, and that the suppression of adipogenic ability is a critical factor for the development of OS.

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Cancer stem cells in malignant glioma - The mechanism of cancer initiation and the therapeutic development

Nakano Ichiro, Saya Hideyuki

Neurological Surgery 38 ( 10 ) 879 - 889 2010.10

ISSN 0301-2603

Malignant glioma is one of the most lethal diseases in adulthood. The median survival of patients with the Grade IV glioma, glioblastoma multiforme (GBM), is shorter than 15 months and the current first-line therapies for this devastating disease have only a palliative effect. The cancer stem cell hypothesis has recently attracted a great deal of attention, owing to the promise of a novel cellular target for the treatment of tumors including GBM. Recent studies have demonstrated that existence of cancer stem cells in brain tumors (BTSC) accounts, at least in part, for the intractability of malignant glioma. From the therapeutic standpoint, characterization of the mechanism for tumor initiation and maintenance of the "stem-like state" of BTSC is crucial. However, multiple heterogeneous subtypes of cancer stem cells have recently been identified from malignant glioma, making the idea of cancer stem cell complicated. In addition, in some cancer types (e.g. melanoma), a considerable proportion of tumor cells may possess the stem cell property, indicating cancer stem cells may not be a rare cell population in tumors, at least in some organs. Based on the extensive genetic and epigenetic characterization of tumor growth mechanisms, various molecularly-targeted therapies have already been applied for patients, demonstrating a varying degree of success in cancer treatment. A significant improvement in patient prognosis was achieved in several cancer types including leukemia and breast cancer. It is no doubt that continuous effort is required to bring hope for patients with malignant glioma. In this study, we summarize the recent findings and approaches in the cancer stem cell field, mainly focusing on malignant glioma stem cells, and also describe potential future directions in this area.

Decreased expression of neurofibromin contributes to epithelial-mesenchymal transition in neurofibromatosis type 1

Arima Yoshimi, Hayashi Hidemi, Kamata Kanako, Goto Takaaki M., Sasaki Mikako, Kuramochi Akira, Saya Hideyuki

Experimental Dermatology 19 ( 8 ) 2010.08

ISSN 0906-6705

Plexiform and/or dermal neurofibromas are nerve sheath tumors of the peripheral nervous system that are usually present in individuals with neurofibromatosis type 1 (NF1). Neurofibromas arise from Schwann cells with biallelic inactivation of NF1, the gene that encodes neurofibromin. This protein is responsible for regulation of the Ras-mediated pathway, which has been shown to play a crucial role in epithelial-to-mesenchymal transition (EMT). EMT is a biological process that occurs during embryogenesis and wound healing and is involved in pathological processes such as organ fibrosis and cancer metastasis. However, the relationship between neurofibromin and EMT has not been elucidated. We investigated whether the EMT-related signaling pathway was upregulated in NF1-associated neurofibromas and Schwann cells by assessing the expression levels of the EMT-related transcription factors Snail, Slug, Twist, ZEB1 and ZEB2. Immunohistochemical studies and quantitative reverse transcription polymerase chain reaction revealed an increase in the expression levels of EMT-related transcription factors in neurofibroma specimens and NF1-derived Schwann cells (sNF96.2). In addition, the silencing of NF1 by siRNA induced the expression of EMT-related transcription factors in normal human Schwann cells and in epithelial-like breast cancer cells. Our findings suggest that the loss of neurofibromin activated the EMT-related signaling pathway and that the excessive mesenchymal reaction may play a key role in the development of NF1-associated neurofibromas.

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The anaphase-promoting complex/cyclosome activator Cdh1 modulates Rho GTPase by targeting p190 RhoGAP for degradation

Naoe Hideaki, Araki Kimi, Nagano Osamu, Kobayashi Yusuke, Ishizawa Jo, Chiyoda Tatsuyuki, Shimizu Takatsune, Yamamura Ken Ichi, Sasaki Yutaka, Saya Hideyuki, Kuninaka Shinji

Molecular and Cellular Biology 30 ( 16 ) 3994 - 4005 2010.08

ISSN 0270-7306

Cdh1 is an activator of the anaphase-promoting complex/cyclosome and contributes to mitotic exit and G₁ maintenance by targeting cell cycle proteins for degradation. However, Cdh1 is expressed and active in postmitotic or quiescent cells, suggesting that it has functions other than cell cycle control. Here, we found that homozygous Cdh1 gene-trapped (Cdh1 ^GT/GT) mouse embryonic fibroblasts (MEFs) and Cdh1-depleted HeLa cells reduced stress fiber formation significantly. The GTP-bound active Rho protein was apparently decreased in the Cdh1-depleted cells. The p190 protein, a major GTPase-activating protein for Rho, accumulated both in Cdh1 ^GT/GT MEFs and in Cdh1-knockdown HeLa cells. Cdh1 formed a physical complex with p190 and stimulated the efficient ubiquitination of p190, both in in vitro and in vivo. The motility of Cdh1-depleted HeLa cells was impaired; however, codepletion of p190 rescued the migration activity of these cells. Moreover, Cdh1^GT/GT embryos exhibited phenotypes similar to those observed for Rho-associated kinase I and II knockout mice: eyelid closure delay and disruptive architecture with frequent thrombus formation in the placental labyrinth layer, respectively. Furthermore, the p190 protein accumulated in the Cdh1^GT/GT embryonic tissues. Our data revealed a novel function for Cdh1 as a regulator of Rho and provided insights into the role of Cdh1 in cell cytoskeleton organization and cell motility.

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Proteome profiling reveals gender differences in the composition of human serum

Miike Koichiro, Aoki Masashi, Yamashita Ryo, Takegawa Yumiko, Saya Hideyuki, Miike Teruhisa, Yamamura Ken Ichi

Proteomics 10 ( 14 ) 2678 - 2691 2010.07

ISSN 1615-9853

Proteome analysis using human serum is a technological advancement that will enable the discovery of novel biomarkers and biomarker patterns of various human diseases. Although proteome analysis using serum has potential in disease prevention, early diagnosis and treatment of diseases, and evaluation of pharmacotherapies, this technology is still in its infancy. Thus, we sought to develop an advanced method of conducting proteome analysis on human serum. In this study, we report the development of the semi-comprehensive protein analytical technique, which involves the systematic use of iTRAQ labeling, HPLC, nano-LC and MS. We compared the composition of the serum proteome in males and females using this technique and detected gender-based differences in serum protein composition. This technology will enable the generation of databases that may ultimately lead to the discovery of specific biomarkers or biomarker patterns of various diseases.

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Breast cancer stem cells

Kai Kazuharu, Arima Yoshimi, Kamiya Toshio, Saya Hideyuki

Breast Cancer 17 ( 2 ) 80 - 85 2010.04

ISSN 1340-6868

Since the initial discovery of leukemia stem cells nearly a decade ago, a great deal of cancer research has focused on the identification of cancer stem cells (CSCs) in many types of solid tumors, including breast cancer. Through analysis of cell surface markers and xenotransplant models, a subpopulation of putative human breast cancer stem cells (BCSCs) that is CD24-negative/CD44- positive (CD24^-/ CD44⁺) and bears high aldehyde dehydrogenase 1 activity has been isolated in clinical samples of breast cancer tissues. Human BCSCs are considered to be derived from basal cells that reside in the basal membranes of alveolar units in human adult mammary glands. Furthermore, BCSCs have been shown to express higher levels of oxidative stress-responsive genes, which could confer part of their ability to resist anti-cancer therapy, than non-CSCs. The emerging picture of the biological properties of BCSCs would contribute for devising innovative therapies for breast cancer, targeting the intrinsic and extrinsic factors that maintain the BCSCs.

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Osteopontin-mediated enhanced hyaluronan binding induces multidrug resistance in mesothelioma cells

Tajima K., Ohashi R., Sekido Y., Hida T., Nara T., Hashimoto M., Iwakami S., Minakata K., Yae T., Takahashi F., Saya H., Takahashi K.

Oncogene 29 ( 13 ) 1941 - 1951 2010.04

ISSN 0950-9232

Malignant pleural mesothelioma (MPM) is resistant to chemotherapy and thus shows a dismal prognosis. Osteopontin (OPN), a secreted noncollagenous and phosphoprotein, is suggested to be involved in the pathogenesis of MPM. However, the precise role of OPN, especially in the multidrug resistance of MPM, remains to be elucidated. We therefore established stable transfectants (ACC-MESO-1/OPN), which constitutively express OPN, to determine its role in the chemoresistance observed in MPM. The introduction of the OPN gene provides MPM cells with upregulated multidrug resistance through the mechanism of enhanced hyaluronate (HA) binding. The expression of CD44 variant isoforms, which inhibit HA binding, significantly decreased in ACC-MESO-1/OPN cells in comparison to control transfectants. Interestingly, the inhibition of the HA-CD44 interaction abrogated multidrug resistance in the ACC-MESO-1/OPN, thus suggesting the involvement of the surviving signal emanating from the HA-CD44 interaction. An enhanced level of the p-Akt in ACC-MESO-1/OPN cells was observed, and was diminished by CD44 siRNA. Inhibition of the Akt phosphorylation increased in number of the cells underwent apoptosis induced by NVB, VP-16 and GEM. Collectively, these results indicate that OPN is strongly involved in multidrug resistance by enhancing the CD44 binding to HA.

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CD44⁺ slow-cycling tumor cell expansion is triggered by cooperative actions of Wnt and prostaglandin E₂ in gastric tumorigenesis

Ishimoto Takatsugu, Oshima Hiroko, Oshima Masanobu, Kai Kazuharu, Torii Ryota, Masuko Takashi, Baba Hideo, Saya Hideyuki, Nagano Osamu

Cancer Science 101 ( 3 ) 673 - 678 2010.03

ISSN 1347-9032

Similar to normal tissue stem cells, cancer stem cells (CSCs) are thought to be quiescent or slow-cycling and, thereby, insensitive to chemo- and radiotherapies. CD44, a cell surface component that interacts with the extracellular matrix, has been found to be highly expressed in CSCs of several solid tumors. However, the relevancy between CD44⁺ cells and slow-cycling cells and the underlying mechanisms for the emergence of CD44⁺ CSCs during tumorigenesis have not been elucidated. Here we show that a gastric gland residing at the squamo-columnar junction (SCJ) in normal mouse stomach contains CD44⁺ stem cell-like slow-cycling cells and that this characteristic CD44⁺ gland was expanded by prostaglandin E2 (PGE₂) and Wnt signaling in K19-Wnt1/C2mE mouse, a genetic mouse model for gastric tumorigenesis. The analysis of three transgenic mouse lines, K19-Wnt1, K19-C2mE and K19-Wnt1/C2mE, revealed that the expansion of CD44⁺ SCJ cells is triggered by PGE₂-mediated signaling and is prominently enhanced by the addition of Wnt activation. Furthermore, each expanded CD44⁺ gland in gastric tumor of K19-Wnt1/C2mE mouse contains a few BrdU label-retaining quiescent or slow-cycling cells, suggesting that the CD44⁺ SCJ cells in normal mouse are candidates for the cell-of-origin of gastric CSCs. These observations suggest that PGE₂-mediated inflammatory signaling and Wnt signaling cooperatively trigger the expansion of CD44⁺ slow-cycling stem-like cells in SCJ, leading to development of lethal gastric tumors in mice.

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Myristoylated alanine-rich C kinase substrate phosphorylation promotes cholangiocarcinoma cell migration and metastasis via the protein kinase C-dependent pathway

Techasen Anchalee, Loilome Watcharin, Namwat Nisana, Takahashi Eri, Sugihara Eiji, Puapairoj Anucha, Miwa Masanao, Saya Hideyuki, Yongvanit Puangrat

Cancer Science 101 ( 3 ) 658 - 665 2010.03

ISSN 1347-9032

Myristoylated alanine-rich C kinase substrate (MARCKS), a substrate of protein kinase C (PKC) has been suggested to be implicated in cell adhesion, secretion, and motility through the regulation of the actin cytoskeletal structure. The quantitative real-time-polymerase chain reaction analysis revealed that MARCKS is significantly overexpressed in Opisthorchis viverrini-associated cholangiocarcinoma (CCA) (P = 0.001) in a hamster model, which correlated with the results of mRNA in situ hybridization. An immunohistochemical analysis of 60 CCA patients revealed a significant increase of MARCKS expression. Moreover, the log-rank analysis indicated that CCA patients with a high MARCKS expression have significantly shorter survival times than those with a low MARCKS expression (P = 0.02). This study investigated whether MARCKS overexpression is associated with CCA metastasis. Using a confocal microscopic analysis of CCA cell lines that had been stimulated with the PKC activator, 12-. 0-tetradecanoyl phorbol-13-acetate (TPA), MARCKS was found to be translocated from the plasma membrane to the perinuclear area. In addition, phosphorylated MARCKS (pMARCKS) became highly concentrated in the perinuclear area. Moreover, an adhesion assay demonstrated that the exogenous overexpression of MARCKS remarkably promoted cell attachment. Interestingly, after TPA stimulation, the CCA cell line-depleted MARCKS showed a decrease in migration and invasion activity. It can be concluded that in non-stimulation, MARCKS promotes cell attachment to the extracellular matrix. After TPA stimulation, PKC phosphorylates MARCKS leading to cell migration or invasion. Taken together, the results of this study reveal a prominent role for MARCKS as one of the key players in the migration of CCA cells and suggest that cycling between MARCKS and pMARCKS can regulate the metastasis of biliary cancer cells.

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Experimental therapies

Imamura Chiyo K., Takebe Naoko, Nakamura Seigo, Saya Hideyuki, Ueno Naoto T.

Nature Reviews Clinical Oncology 7 ( 3 ) 127 - 128 2010.03

ISSN 1759-4774

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Tumor necrosis factor-α regulates transforming growth factor-β-dependent epithelial-mesenchymal transition by promoting hyaluronan-CD44-moesin interaction

Takahashi Eri, Nagano Osamu, Ishimoto Takatsugu, Yae Toshifumi, Suzuki Yoshimi, Shinoda Takeshi, Nakamura Satoshi, Niwa Shinichiro, Ikeda Shun, Koga Hisashi, Tanihara Hidenobu, Saya Hideyuki

Journal of Biological Chemistry 285 ( 6 ) 4060 - 4073 2010.02

ISSN 0021-9258

Aberrant epithelial-mesenchymal transition (EMT) is involved in development of fibrotic disorders and cancer invasion. Alterations of cell-extracellular matrix interaction also contribute to those pathological conditions. However, the functional interplay between EMT and cell-extracellular matrix interactions remains poorly understood.Wenow show that the inflammatory mediator tumor necrosis factor-α (TNF-α) induces the formation of fibrotic foci by cultured retinal pigment epithelial cells through activation of transforming growth factor-β (TGF-β) signaling in a manner dependent on hyaluronan-CD44-moesin interaction. TNF-α promoted CD44 expression and moesin phosphorylation by protein kinase C, leading to the pericellular interaction of hyaluronan and CD44. Formation of the hyaluronan-CD44-moesin complex resulted in both cell-cell dissociation and increased cellular motility through actin remodeling. Furthermore, this complex was found to be associated with TGF-β receptor II and clathrin at actin microdomains, leading to activation of TGF-β signaling. We established an in vivo model of TNF-α-induced fibrosis in the mouse eye, and such ocular fibrosis was attenuated in CD44-null mice. The production of hyaluronan and its interaction with CD44, thus, play an essential role in TNF-α-induced EMT and are potential therapeutic targets in fibrotic disorders.

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PSF1, a DNA replication factor expressed widely in stem and progenitor cells, drives tumorigenic and metastatic properties

Nagahama Yumi, Ueno Masaya, Miyamoto Satoru, Morii Eiichi, Minami Takashi, Mochizuki Naoki, Saya Hideyuki, Takakura Nobuyuki

Cancer Research 70 ( 3 ) 1215 - 1224 2010.02

ISSN 0008-5472

PSF1 (partner of sld five 1) is an evolutionarily conserved DNA replication factor implicated in DNA replication in lower species that is strongly expressed in a wide range of normal stem cell populations and progenitor cell populations. Because stem and progenitor cells possess high proliferative capacity, we hypothesized that PSF1 may play an important role in tumor growth. To begin to investigate PSF1 function in cancer cells, we cloned the mouse PSF1 promoter and generated lung and colon carcinoma cells that stably express a PSF1 promoter-reporter gene. Reporter expression in cells correlated with endogenous PSF1 mRNA expression. In a tumor cell xenograft model, high levels of reporter expression correlated with high proliferative activity, serial transplantation potential, and metastatic capability. Notably, cancer cells expressing reporter levels localized to perivascular regions in tumors and displayed expression signatures related to embryonic stem cells. RNAi-mediated silencing of endogenous PSF1 inhibited cancer cell growth by disrupting DNA synthesis and chromosomal segregation. These findings implicate PSF1 in tumorigenesis and offer initial evidence of its potential as a theranostic target.

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Molecularly targeted therapies for glioma

Yamanaka Ryuya, Saya Hideyuki

Annals of Neurology 66 ( 6 ) 717 - 729 2009.12

ISSN 0364-5134

Over the past decade, molecularly targeted therapies have been added to cytotoxic and antiendocrine drugs in the treatment of cancer, with the aim of targeting the molecular pathways that underlie the carcinogenic process and maintain the cancer phenotype. Success with some of these agents has suggested that identification and validation of drug targets is the starting point for the development of active, safe, and effective drugs. The main molecular targets used to develop anticancer drugs are cell surface receptors, signal transduction pathways, gene transcription targets, ubiquitin-proteasome/heat shock proteins, and tumor micro-environment components. Here, we review the development of the main molecularly targeted noncytotoxic agents studied in glioma, highlighting lessons derived from the development of these novel drugs and proposing new horizons for the clinical development of molecularly targeted therapies.

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Maintenance of HCT116 colon cancer cell line conforms to a stochastic model but not a cancer stem cell model

Kai Kazuharu, Nagano Osamu, Sugihara Eiji, Arima Yoshimi, Sampetrean Oltea, Ishimoto Takatsugu, Nakanishi Masaya, Ueno Naoto T., Iwase Hirotaka, Saya Hideyuki

Cancer Science 100 ( 12 ) 2275 - 2282 2009.12

ISSN 1347-9032

The cancer stem cell (CSC) model, in which a small population of cells within a tumor possesses the ability to self-renew and reconstitute the phenotype of primary tumor, has gained wide acceptance based on evidence over the past decade. It has also been reported that cancer cell lines contain a CSC subpopulation. However, phenotypic differences between CSCs and non-CSCs in cancer cell lines are not better defined than in primary tumors. Furthermore, some cell lines do not have a CSC population, revealed as a side population and expression of CD133. Thus, the identification of CSCs in cancer cell lines remains elusive. Here, we investigated the CSC hierarchy within HCT116 colon cancer cells, which do not have a CD133-positive subpopulation. We examined the expression of alternative CSC markers epithelial specific antigen (ESA) and CD44 in floating-sphere-derived cells, which are known to be the cells of enriching CSCs. Sphere-derived HCT116 cells exhibited heterogeneous expression of ESA and CD44. The two major subpopulations of HCT116 sphere cells (ESA^lowCD44^-/low and ESA^highCD44^high) exhibited a biological/proliferative hierarchy of sphere-forming and soft agar colony-forming activity. However, there was no difference between the two subpopulations in the incidence of xenograft tumors. When ESA^lowCD44^-/low cells were allowed to aggregate and re-form floating-spheres, the biological/proliferative hierarchy of parental HCT116 spheres was reconstituted, in terms of ESA and CD44 expression. Thus, HCT116 cells have plasticity when they are set in floating-spheres, suggesting that maintenance of the HCT116 cell line conforms to a stochastic model, not a CSC model.

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Identification of tumor-initiating cells in a highly aggressive brain tumor using promoter activity of nucleostemin

Tamase Akira, Muraguchi Teruyuki, Naka Kazuhito, Tanaka Shingo, Kinoshita Masashi, Hoshii Takayuki, Ohmura Masako, Shugo Haruhiko, Ooshio Takako, Nakada Mitsutoshi, Sawamoto Kazunobu, Onodera Masafumi, Matsumoto Kunio, Oshima Masanobu, Asano Masahide, Saya Hideyuki, Okano Hideyuki, Suda Toshio, Hamada Jun Ichiro, Hirao Atsushi

Proceedings of the National Academy of Sciences of the United States of America 106 ( 40 ) 17163 - 17168 2009.10

ISSN 0027-8424

Controversy remains over whether the cancer stem cell (CSC) theory applies to all tumors. To determine whether cells within a highly aggressive solid tumor are stochastically or hierarchically organized, we combined a reporter system where the nucleostemin (NS) promoter drives GFP expression (termed NS-GFP) with a mouse brain tumor model induced by retroviral Ras expression on a p16 ^Ink4a/p19^Arf-deficient background. The NS-GFP system allowed us to monitor the differentiation process of normal neural stem/precursor cells by analyzing GFP fluorescence intensity. In tumor-bearing mice, despite the very high frequency of tumorigenic cells, we successfully identified the NS-GFP⁺ cells as tumorinitiating cells (T-ICs). The clonal studies conclusively established that phenotypical heterogeneity can exist among the cells comprising a genetically homogeneous tumor, suggesting that this aggressive brain tumor follows the CSC model. Detailed analyses of the NS-GFP⁺ brain tumor cells revealed that T-ICs showed activation of the receptor tyrosine kinase c-Met, which functions in tumor invasiveness. Thus, the NS-GFP system provides a powerful tool to elucidate stem cell biology in normal and malignant tissues.

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Current clinical trials using new targeted therapies for myeloid leukemia and the research trends

Ishizawa J., Saya Hideyuki

Nippon rinsho. Japanese journal of clinical medicine 67 ( 10 ) 1932 - 1937 2009.10

ISSN 0047-1852

Great progress on insight into genetic aberrations of myeloid leukemia via gene expression profiling has led to better understanding of the pathobiology of this heterogeneous disorder. It enabled the development of specific treatment modalities targeted to underlying oncogenic abnormalities, with well established examples of all-trans retinoic acid for the treatment of acute promyelocytic leukemia and imatinib for chronic myeloid leukemia. However, these strategies have not been completely developed yet in that most of brand new targeted therapies have been somewhat far from achieving cure of leukemia and that many problems with regards to drug resistance and recurrence from minimal residual disease remain to be solved. On the other hand, concept of cancer(leukemic) stem cell and its niche has been shedding new light on oncological field these days. This review summarizes the current clinical trials using new targeted therapies and research trends on myeloid leukemia.

Simian virus 40 large T antigen targets the microtubule-stabilizing protein TACC2

Tei Shuchin, Saitoh Noriko, Funahara Tetsushi, Iida Shin Ichi, Nakatsu Yuko, Kinoshita Kayo, Kinoshita Yoshikazu, Saya Hideyuki, Nakao Mitsuyoshi

Journal of Cell Science 122 ( 17 ) 3190 - 3198 2009.09

ISSN 0021-9533

The large T antigens of polyomaviruses target cellular proteins that control fundamental processes, including p53 and the RB family of tumor suppressors. Mechanisms that underlie Tantigen- induced cell transformation need to be fully addressed, because as-yet unidentified target proteins might be involved in the process. In addition, recently identified polyomaviruses are associated with particular human diseases such as aggressive skin cancers. Here, we report that simian virus 40 (SV40) large T antigen interacts with the transforming acidic coiled-coilcontaining protein TACC2, which is involved in stabilizing microtubules in mitosis. T antigen directly binds TACC2 and induces microtubule dysfunction, leading to disorganized mitotic spindles, slow progression of mitosis and chromosome missegregation. These mitotic defects are caused by N-terminaldeleted T antigen, which minimally interacts with TACC2, whereas T-antigen-induced microtubule destabilization is suppressed by overexpressing TACC2. Thus, TACC2 might be a key target of T antigen to disrupt microtubule regulation and chromosomal inheritance in the initiation of cell transformation.

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Real-time in vivo imaging of p16^Ink4a reveals cross talk with p53

Yamakoshi Kimi, Takahashi Akiko, Hirota Fumiko, Nakayama Rika, Ishimaru Naozumi, Kubo Yoshiaki, Mann David J., Ohmura Masako, Hirao Atsushi, Saya Hideyuki, Arase Seiji, Hayashi Yoshio, Nakao Kazuki, Matsumoto Mitsuru, Ohtani Naoko, Hara Eiji

Journal of Cell Biology 186 ( 3 ) 393 - 407 2009.08

ISSN 0021-9525

Expression of the p16^Ink4a tumor suppressor gene, a sensor of oncogenic stress, is up-regulated by a variety of potentially oncogenic stimuli in cultured primary cells. However, because p16^Ink4a expression is also induced by tissue culture stress, physiological mechanisms regulating p16^Ink4a expression remain unclear. To eliminate any potential problems arising from tissue culture-imposed stress, we used bioluminescence imaging for noninvasive and real-time analysis of p16^Ink4a expression under various physiological conditions in living mice. In this study, we show that oncogenic insults such as ras activation provoke epigenetic derepression of p16^Ink4a expression through reduction of DNMT1 (DNA methyl transferase 1) levels as a DNA damage response in vivo. This pathway is accelerated in the absence of p53, indicating that p53 normally holds the p16^Ink4a response in check. These results unveil a backup tumor suppressor role for p16^Ink4a in the event of p53 inactivation, expanding our understanding of how p16^Ink4a expression is regulated in vivo.

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BubR1 localizes to centrosomes and suppresses centrosome amplification via regulating Plk1 activity in interphase cells

Izumi H., Matsumoto Y., Ikeuchi T., Saya H., Kajii T., Matsuura S.

Oncogene 28 ( 31 ) 2806 - 2820 2009.08

ISSN 0950-9232

BubR1 is a critical component of the mitotic checkpoint that delays the onset of anaphase until all chromosomes have established bipolar attachment to the microtubules. We previously reported that mutations of the BUB1B gene (encoding BubR1) caused premature chromatid separation (PCS) syndrome, a condition characterized by constitutional aneuploidy and a high risk of childhood cancer. We here report that the cells from PCS syndrome patients have loss of regulation of the centrosome duplication machinery, resulting in centrosome amplification and multipolar mitosis. PCS syndrome cells show increased activity of Polo-like kinase 1 (Plk1), whose knockdown suppresses centrosome amplification. BubR1 localizes to centrosomes, physically interacts with Plk1 and inhibits Plk1 phosphorylation and its kinase activity during interphase. These results unravel a crucial role of BubR1 in preventing centrosome reduplication through negative regulation of Plk1 in interphase cells.

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Molecular mechanism of cell death induced by chemotherapeutic agents

Saya Hideyuki

Brain and Nerve 61 ( 7 ) 843 - 847 2009.07

ISSN 1881-6096

Cells maintain their homeostasis by checkpoint mechanisms that exist at each phase of the cell cycle. Impairment of the checkpoint mechanisms induces DNA mutation and asymmetric segregation of chromosomes, which result in genomic instability, leading to malignant cell transformation. DNA-damaging agents, which are still the first-line chemotherapeutic drugs for various cancers, have recently been demonstrated to induce death in cancer cells alone just because these cells have abnormalities in their checkpoint mechanisms. The mitotic checkpoint, in particular, has been found to play a key role in the drug-induced cell death. An understanding of the molecular mechanisms of chemotherapy will enable us to use conventional drugs more appropriately for cancer treatment.

Overexpression of Aurora A by loss of CHFR gene expression increases the growth and survival of HTLV-1-infected T cells through enhanced NF-κB activity

Tomita Mariko, Toyota Minoru, Ishikawa Chie, Nakazato Tetsuro, Okudaira Taeko, Matsuda Takehiro, Uchihara Jun Nosuke, Taira Naoya, Ohshiro Kazuiku, Senba Masachika, Tanaka Yuetsu, Ohshima Koichi, Saya Hideyuki, Tokino Takashi, Mori Naoki

International Journal of Cancer 124 ( 11 ) 2607 - 2615 2009.06

ISSN 0020-7136

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent for adult T-cell leukemia (ATL). Aurora A, a mitotic checkpoint protein, is overexpressed in human cancer cells. The cell cycle-dependent turnover of Aurora A is regulated by E3 ubiquitin ligases such as checkpoint with fork head-associated and ring finger (CHFR). Here, we found overexpression of Aurora A protein in HTLV-1-infected T-cell lines and primary ATL cells. The expression of CHFR mRNA was reduced in these cells by abnormal methylation of CHFR promoter region. Knockdown of Aurora A using small interfering RNA suppressed the growth of HTLV-1-infected T-cell line. Transfection of Aurora A expression plasmid enhanced Tax-induced nuclear factor-κB (NF-κB) reporter activity. Transfection of CHFR expression plasmid into an HTLV-1-infected T-cell line reduced cell growth, Aurora A protein level and constitutive NF-κB reporter activity. Aurora kinase inhibitor suppressed the growth and survival of HTLV-1-infected T-cell lines and primary ATL cells. It also reduced constitutive NF-κB activity in an HTLV-1-infected T-cell line by reducing IκB kinase κ phosphorylation and the expression of antiapoptotic protein survivin. Our results suggested that loss of CHFR expression resulted to accumulation of Aurora A, which increased NF-κB activity. These findings highlight the critical role of Aurora A in HTLV-1-infected T cells, making this molecule a potentially suitable target for future therapies for ATL.

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M-CSF inhibition selectively targets pathological angiogenesis and lymphangiogenesis

Kubota Yoshiaki, Takubo Keiyo, Shimizu Takatsune, Ohno Hiroaki, Kishi Kazuo, Shibuya Masabumi, Saya Hideyuki, Suda Toshio

Journal of Experimental Medicine 206 ( 5 ) 1089 - 1102 2009.05

ISSN 0022-1007

Antiangiogenic therapy for the treatment of cancer and other neovascular diseases is desired to be selective for pathological angiogenesis and lymphangiogenesis. Macrophage colony-stimulating factor (M-CSF), a cytokine required for the differentiation of monocyte lineage cells, promotes the formation of high-density vessel networks in tumors and therefore possesses therapeutic potential as an M-CSF inhibitor. However, the physiological role of M-CSF in vascular and lymphatic development, as well as the precise mechanisms underlying the antiangiogenic effects of M-CSF inhibition, remains unclear. Moreover, therapeutic potential of M-CSF inhibition in other neovascular diseases has not yet been evaluated. We used osteopetrotic (op/op) mice to demonstrate that M-CSF deficiency reduces the abundance of LYVE-1⁺ and LYVE1^- macrophages, resulting in defects in vascular and lymphatic development. In ischemic retinopathy, M-CSF was required for pathological neovascularization but was not required for the recovery of normal vasculature. In mouse osteosarcoma, M-CSF inhibition effectively suppressed tumor angiogenesis and lymphangiogenesis, and it disorganized extracellular matrices. In contrast to VEGF blockade, interruption of M-CSF inhibition did not promote rapid vascular regrowth. Continuous M-CSF inhibition did not affect healthy vascular and lymphatic systems outside tumors. These results suggest that M-CSF-targeted therapy is an ideal strategy for treating ocular neovascular diseases and cancer.

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K858, a novel inhibitor of mitotic kinesin Eg5 and antitumor agent, induces cell death in cancer cells

Nakai Ryuichiro, Iida Shin Ichi, Takahashi Takeshi, Tsujita Tetsuya, Okamoto Seiho, Takada Chie, Akasaka Kazuhito, Ichikawa Shunji, Ishida Hiroyuki, Kusaka Hideaki, Akinaga Shiro, Murakata Chikara, Honda Shinobu, Nitta Masayuki, Saya Hideyuki, Yamashita Yoshinori

Cancer Research 69 ( 9 ) 3901 - 3909 2009.05

ISSN 0008-5472

The aim of this study was to investigate the mechanism of inhibition of Eg5 (kinesin spindle protein), a mitotic kinesin that plays an essential role in establishing mitotic spindle bipolarity, by the novel small molecule inhibitor K858. K858 was selected in a phenotype-based forward chemical genetics screen as an antimitotic agent, and subsequently characterized as an inhibitor of Eg5. K858 blocked centrosome separation, activated the spindle checkpoint, and induced mitotic arrest in cells accompanied by the formation of monopolar spindles. Long-term continuous treatment of cancer cells with K858 resulted in antiproliferative effects through the induction of mitotic cell death, and polyploidization followed by senescence. In contrast, treatment of nontransformed cells with K858 resulted in mitotic slippage without cell death, and cell cycle arrest in G₁ phase in a tetraploid state. In contrast to paclitaxel, K858 did not induce the formation of micronuclei in either cancer or nontransformed cells, suggesting that K858 has minimal effects on abnormalities in the number and structure of chromosomes. K858 exhibited potent antitumor activity in xenograft models of cancer, and induced the accumulation of mitotic cells with monopolar spindles in tumor tissues. Importantly, K858, unlike antimicrotubule agents, had no effect on microtubule polymerization in cell-free and cellbased assays, and was not neurotoxic in a motor coordination test in mice. Taken together, the Eg5 inhibitor K858 represents an important compound for further investigation as a novel anticancer therapeutic.

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[Mitotic regulation and carcinogenesis].

Saya Hideyuki, Zhang Dongwei, Oltea Sampetrean

Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 54 ( 4 Suppl ) 561 - 567 2009.03

ISSN 0039-9450

Reversible whole-organism cell cycle arrest in a living vertebrate

Sampetrean Oltea, Lida Shin Ichi, Makino Shinji, Matsuzaki Yuriko, Ohno Kikuo, Saya Hideyuki

Cell Cycle 8 ( 4 ) 620 - 627 2009.02

ISSN 1538-4101

In vivo cell cycle analysis in higher eukaryotes has been limited by the challenge of preserving the integrity of the living organism while visualizing dividing cells. Japanese medaka in order to visualize and manipulate the cell cycle progression in a live vertebrate. Our stable transgenic histone H2B-GFP medaka line allows fluorescence-based monitoring of the chromosomes. The system has a high specificity, with a strong GFP signal labeling the chromatin architecture. The subcellular resolution ensures detection of both normal and abnormal divisions in live recordings. This translates into the possibility to quantify temporal and spatial aspects of the cell cycle, such as length or nuclear size, as well as to expose drug toxicity at the earliest stage. We also show that acclimation to cold, a prominent feature of the eurytherm medaka, is a valuable natural way of inducing a reversible cell cycle arrest in the entire living organism. Our results suggest that this manipulation can be performed from the early stages of development, has no toxicity and does not alter the cell cycle profile of the embryo.

Molecular mechanism regulating effect of anti-cancer agents

Saya Hideyuki

Japanese Journal of Cancer and Chemotherapy 36 ( 1 ) 1 - 5 2009.01

ISSN 0385-0684

Faithful genome duplication is achieved by accurate coordination between DNA replication and chromosome segregation. Abnormalities occurring in this process are checked by biochemical signal transduction pathways, called checkpoints, which ensure the orderly progression of events in the cell cycle. Checkpoints prevent transition into subsequent phases until all processes in the previous phase are completed. Defects in cell cycle checkpoints result in gene mutations, chromosome damage, and aneuploidy, all of which contribute to tumorigenesis. However, it has recently been uncovered that the impairment of checkpoint function is the major reason why DNA damaging anti-cancer agents can selectively kill cancer cells. Given that G₁ and G₂ checkpoint functions are generally impaired in cancer cells, cells with DNA damage are unable to maintain G₂ arrest and eventually die as they enter mitosis. This process is known as mitotic catastrophe.

Molecular mechanisms regulating dissociation of cell-cell junction of epithelial cells by oxidative stress

Inumaru Junko, Nagano Osamu, Takahashi Eri, Ishimoto Takatsugu, Nakamura Satoshi, Suzuki Yoshimi, Niwa Shin Ichiro, Umezawa Kazuo, Tanihara Hidenobu, Saya Hideyuki

Genes to Cells 14 ( 6 ) 703 - 716 2009

ISSN 1356-9597

Oxidative stress is regarded as a causative factor in aging and various degenerative diseases. Here, we show the mechanism by which oxidative stress induces disruption of cell-cell junctions using retinal pigment epithelial cells. We demonstrated that reactive oxygen species (ROS)-mediated activation of Src kinase increases the tyrosine phosphorylation state of p120-catenin and rapidly triggers translocation of p120-catenin and internalization of N-cadherin from the cell-cell adhesion sites to an early endosomal compartment. Endosomal accumulation of p120-catenin resulted in stress fiber formation and cell-cell dissociation through the activation of Rho/Rho kinase pathway. However, these cytoskeletal remodeling and cell-cell dissociation induced by oxidative stress were transient, due to the activation of nuclear factor-κB (NF-κB) and the expression of manganese superoxide dismutase (Mn-SOD). Using the NF-κB specific inhibitor DHMEQ, we found that NF-κB is part of a negative feedback loop to control intracellular ROS levels. Finally, we demonstrated that H₂ O₂ treatment alone does not induce the epithelial mesenchymal transition (EMT) in retinal pigment epithelial cells, which can be induced by TNF-α treatment. These findings suggest that oxidative stress is a crucial factor to induce the cell-cell dissociation, an initial step of EMT, but does not provide sufficient signals to establish and to maintain the EMT.

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Brain tumor stem cells from an adenoid glioblastoma multiforme

Saya Hideyuki

Neurologia Medico-Chirurgica 49 ( 4 ) 151 2009

ISSN 0470-8105

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Involvement of hyaluronan and its receptor CD44 with choroidal neovascularization

Mochimaru Hiroshi, Takahashi Eri, Tsukamoto Nobuo, Miyazaki Junichiro, Yaguchi Tomonori, Koto Takashi, Kurihara Toshihide, Noda Kousuke, Ozawa Yoko, Ishimoto Takatsugu, Kawakami Yutaka, Tanihara Hidenobu, Saya Hideyuki, Ishida Susumu, Tsubota Kazuo

Investigative Ophthalmology and Visual Science 50 ( 9 ) 4410 - 4415 2009

ISSN 0146-0404

PURPOSE. CD44 is a cell-surface adhesion molecule and receptor for hyaluronan (HA), one of the major extracellular matrix components. The purpose of the present study was to clarify a role of HA and CD44 in the development of choroidal neovascularization (CNV). METHODS. Laser photocoagulation was used to induce CNV in C57BL/6 mice or CD44-deficient mice. The mRNA expression of CD44 and HA synthase (HAS)-2 in the retinal pigment epithelium (RPE)-choroid complex was evaluated by DNA microarray and real-time RT-PCR analyses 3 days after laser treatment. HA synthesis and CD44 expression were examined by immunohistochemistry 1 week after photocoagulation. Mice with laser-induced CNV were systemically administered the HA synthesis inhibitor 4-methylumbelliferone (MU) or an anti- CD44-neutralizing antibody. The response of CNV was analyzed by volumetric measurements 1 week after photocoagulation. Macrophage infiltration into CNV lesions was evaluated by real-time RT-PCR for F4/80 3 days after laser-induced injury. RESULTS. The induction of CNV led to a significant increase in expression of CD44 and HAS2 mRNA. HA and CD44 were immunopositive in the CNV lesions. Compared with vehicle treatment, the systemic application of MU significantly attenuated CNV volume in a dose-dependent fashion, together with macrophage infiltration into the lesions. Consistently, antibody- based blockade of CD44 resulted in a significant reduction of CNV volume, compared with the isotype control. In contrast, genetic ablation of CD44 significantly augmented CNV formation together with HA accumulation and macrophage infiltration, compared with wild-type mice. CONCLUSIONS. These results indicate a significant role of HA and its receptor CD44 in the development of CNV. (Invest Ophthalmol Vis Sci.

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An essential role of the aPKC-Aurora A-NDEL1 pathway in neurite elongation by modulation of microtubule dynamics

Mori Daisuke, Yamada Masami, Mimori-Kiyosue Yuko, Shirai Yasuhito, Suzuki Atsushi, Ohno Shigeo, Saya Hideaki, Wynshaw-Boris Anthony, Hirotsune Shinji

Nature Cell Biology 11 ( 9 ) 1057 - 1068 2009

ISSN 1465-7392

Orchestrated remodelling of the cytoskeketon is prominent during neurite extension. In contrast with the extensive characterization of actin filament regulation, little is known about the dynamics of microtubules during neurite extension. Here we identify an atypical protein kinase C (aPKC)-Aurora A-NDEL1 pathway that is crucial for the regulation of microtubule organization during neurite extension. aPKC phosphorylates Aurora A at Thr 287 (T287), which augments interaction with TPX2 and facilitates activation of Aurora A at the neurite hillock, followed by phosphorylation of NDEL1 at S251 and recruitment. Suppression of aPKC, Aurora A or TPX2, or disruption of Ndel1, results in severe impairment of neurite extension. Analysis of microtubule dynamics with a microtubule plus-end marker revealed that suppression of the aPKC-Aurora A-NDEL1 pathway resulted in a significant decrease in the frequency of microtubule emanation from the microtubule organizing centre (MTOC), suggesting that Aurora A acts downstream of aPKC. These findings demonstrate a surprising role of aPKC-Aurora A-NDEL1 pathway in microtubule remodelling during neurite extension.

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p53 regulates toll-like receptor 3 expression and function in human epithelial cell lines

Taura Manabu, Eguma Ayaka, Suico Mary Ann, Shuto Tsuyoshi, Koga Tomoaki, Komatsu Kensei, Komune Takefumi, Sato Takashi, Saya Hideyuki, Li Jian Dong, Kai Hirofumi

Molecular and Cellular Biology 28 ( 21 ) 6557 - 6567 2008.11

ISSN 0270-7306

Toll-like receptors (TLRs) are important sensors of microbial pathogens and mediators of innate immune responses. Although the signal transduction of TLRs is well elucidated, their basal regulation is largely unexplored. Here we show that the tumor suppressor p53 positively regulates the transcription of TLR3, a receptor for viral double-stranded RNA and poly(I-C), by binding to the p53 site in the TLR3 promoter. TLR3 expression was lower in HCT116 p53^-/- cells than in HCT116 p53^+/+ cells. Activation of p53 by 5-fluorouracil increased the TLR3 mRNA in epithelial cell lines with wild-type p53 but not in cell lines harboring mutant p53. Knockdown of p53 by small interfering RNA decreased the TLR3 expression. TLR3 mRNA was also lower in liver and intestine of p53^-/- mice than in p53^-/- mice. Furthermore, the poly(I-C)-induced phosphorylation of IκB-α, nuclear translocation of NF-κB, and phosphorylation of interferon regulatory transcription factor 3, were drastically reduced in HCT116 p53^-/- cells, indicating a dysregulation of the two signaling pathways governed by TLR3. Consequently, induction of interleukin-8 and beta interferon after poly(I-C) stimulation was impaired in HCT116 p53^-/- cells. These results suggest that p53 influences TLR3 expression and function and highlight a role of p53 in innate immune response in epithelial cells.

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An evolutionarily conserved leucine-rich repeat protein CLERC is a centrosomal protein required for spindle pole integrity

Muto Yoshinori, Yoshioka Takashi, Kimura Masashi, Matsunami Miki, Saya Hideyuki, Okano Yukio

Cell Cycle 7 ( 17 ) 2738 - 2748 2008.09

ISSN 1538-4101

The centrosome functions as the microtubule-organizing center (MTOC) and plays a vital role in organizing spindle poles during mitosis. The pair of centrioles, which are the core components of the centrosome, duplicate once per cell cycle, and this feature is essential for the establishment of spindle bipolarity. Here we describe the molecular characterization of a novel protein called CLERC (Centrosomal leucine-rich repeat and coiled-coil containing protein) which is a human ortholog of Chlamydomonas Vfl1 protein. CLERC is a protein of 1032 amino acids with a calculated molecular mass of 120 kDa and possesses leucine-rich repeat and coiled-coil domains. Database searches revealed that CLERC has homologs in a wide variety of eukaryotes and is evolutionarily conserved. Endogenous CLERC protein associated with the centrosomes throughout the cell cycle and accumulated during mitosis. RNAi-mediated depletion of CLERC blocked formation of normal mitotic spindles and led to multipolar spindles. Moreover, many of the spindle poles in CLERC depleted cells contained only one centriole, indicating that centrosomes split into fractions containing a single centriole. These data indicate that the major function of CLERC during mitosis is to maintain the structural integrity of centrosomes, thereby contributing to spindle bipolarity.

Role of CD44-mediated signal in cancer invasion and metastasis

Saya Hideyuki, Nagano Osamu

Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 53 ( 10 ) 1225 - 1230 2008.08

ISSN 0039-9450

AIP regulates stability of Aurora-A at early mitotic phase coordinately with GSK-3β

Fumoto K., Lee P. C., Saya H., Kikuchi A.

Oncogene 27 ( 32 ) 4478 - 4487 2008.07

ISSN 0950-9232

Glycogen synthase kinase-3 (GSK-3β) regulates microtubule dynamics and cellular polarity through phosphorylating various microtubule associating proteins and plus-end tracking proteins. Although it was also reported that GSK-3β is inactivated by protein kinase B at the spindle poles, functions and targets of GSK-3β in the mitotic phase are unknown. Here, we identified Aurora-A-interacting protein (AIP), a negative regulator of Aurora-A, as a binding partner of GSK-3β. AIP was colocalized with Aurora-A and GSK-3β to the spindle poles in metaphase, and its depletion in cells stabilized and activated Aurora-A in early mitotic phase and caused mitotic cell arrest. Treatment of the cells with a GSK-3β inhibitor reduced the protein level of Aurora-A and this reduction was suppressed by AIP knockdown. AIP was phosphorylated by GSK-3β, and an AIP mutant in which the GSK-3β phosphorylation site was mutated could bind and downregulate Aurora-A more efficiently. These results suggest that GSK-3β modulates the early mitotic Aurora-A level through binding and phosphorylating AIP.

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Aurora A overexpression induces cellular senescence in mammary gland hyperplastic tumors developed in p53-deficient mice

Zhang D., Shimizu T., Araki N., Hirota T., Yoshie M., Ogawa K., Nakagata N., Takeya M., Saya H.

Oncogene 27 ( 31 ) 4305 - 4314 2008.07

ISSN 0950-9232

Aurora A mitotic kinase is frequently overexpressed in various human cancers and is widely considered to be an oncoprotein. However, the cellular contexts in which Aurora A induces malignancy in vivo are still unclear. We previously reported a mouse model in which overexpression of human Aurora A in the mammary gland leads to small hyperplastic changes but not malignancy because of the induction of p53-dependent apoptosis. To study the additional factors required for Aurora A-associated tumorigenesis, we generated a new Aurora A overexpression mouse model that lacks p53. We present evidence here that Aurora A overexpression in primary mouse embryonic fibroblasts (MEFs) that lack p53 overrides postmitotic checkpoint and leads to the formation of multinucleated polyploid cells. Induction of Aurora A overexpression in the mammary glands of p53-deficient mice resulted in development of precancerous lesions that were histologically similar to atypical ductal hyperplasia in human mammary tissue and showed increased cellular senescence and p16 expression. We further observed DNA damage in p53-deficient primary MEFs after Aurora A overexpression. Our results suggest that Aurora A overexpression in mammary glands is insufficient for the development of malignant tumors in p53-deficient mice because of the induction of cellular senescence. Both p53 and p16 are critical in preventing mammary gland tumorigenesis in the Aurora A overexpression mouse model.

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Rb depletion results in deregulation of E-cadherin and induction of cellular phenotypic changes that are characteristic of the epithelial-to- mesenchymal transition

Arima Yoshimi, Inoue Yasumichi, Shibata Tatsuhiro, Hayashi Hidemi, Nagano Osamu, Saya Hideyuki, Taya Yoichi

Cancer Research 68 ( 13 ) 5104 - 5112 2008.07

ISSN 0008-5472

The retinoblastoma tumor suppressor protein (Rb) is mutated or expressed at very low levels in several tumor types, including retinoblastoma and osteosarcoma, as well as small cell lung, colon, prostate, bladder, and breast carcinomas. Loss or reduction of Rb expression is seen most commonly in high-grade breast adenocarcinomas, suggesting that a relationship may exist between loss of Rb function and a less-differentiated state, increased proliferation, and high metastatic potential. In this study, we found that knockdown of Rb by small interfering RNA in MCF7 breast cancer cells disrupts cell-cell adhesion and induces a mesenchymal-like phenotype. The epithelial-to-mesenchymal transition (EMT), a key event in embryonic morphogenesis, is implicated in the metastasis of primary tumors. Additionally, Rb is decreased during growth factor- and cytokine-induced EMT and overexpression of Rb inhibits the EMT in MCF10A human mammary epithelial cells. Ectopic expression and knockdown of Rb resulted in increased or reduced expression of E-cadherin, which is specifically involved in epithelial cell-cell adhesion. Other EMT-related transcriptional factors, including Slug and Zeb-1, are also induced by Rb depletion. Furthermore, we confirmed that Rb binds to an E-cadherin promoter sequence in association with the transcription factor activator protein-2A. Finally, in breast cancer specimens, we observed a concurrent downregulation of Rb and E-cadherin expression in mesenchymal-like invasive cancers. These findings suggest that Rb inactivation contributes to tumor progression due to not only loss of cell proliferation control but also conversion to an invasive phenotype and that the inhibition of EMT is a novel tumor suppressor function of Rb.

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The ERK-RSK1 activation by growth factors at G2 phase delays cell cycle progression and reduces mitotic aberrations

Nam Hyun Ja, Kim Sujeong, Lee Min Woo, Lee Bok Soon, Hara Toshihiro, Saya Hideyuki, Cho Hyeseong, Lee Jae Ho

Cellular Signalling 20 ( 7 ) 1349 - 1358 2008.07

ISSN 0898-6568

Growth factors accelerate G0 to S progression in the cell cycle, however, the roles of growth factors in other cell cycle phases are largely unknown. Here, we show that treatment of HeLa cells with hepatocyte growth factor (HGF) at G2 phase induced the G2/M transition delay as evidenced by FACS analysis as well as by mitotic index and time-lapse analyses. Growth factors such as epidermal growth factor (EGF) and fibroblast growth factor (FGF) also induced G2/M transition delay like HGF. HGF treatment at G2 phase causes a delayed activation of cyclin B1-associated kinase and a diminished nuclear translocation of cyclin B1. Either U0126, a MAPK kinase (MEK) inhibitor, or kinase-dead mutant of ribosomal S6 kinase (RSK) abolished the delay. Additionally, knockdown of RSK1, but not RSK2, with siRNA abrogated the delay, indicating that the extracellular-regulated protein kinase (ERK)-RSK1 mediates the HGF-induced delay. We further found that the delay in G2/M transition of cells expressing oncogenic HGF receptor, M1268T, was abolished by RSK1 knockdown. Intriguingly, we observed that HGF induced chromosomal segregation defects, and depletion of RSK1, but not RSK2, aggravated these chromosomal aberrations. Taken together, the ERK-RSK1 activation by growth factors delays G2/M transition and this might be required to maintain genomic integrity during growth factor stimulation.

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Activated macrophages promote Wnt signalling through tumour necrosis factor-α in gastric tumour cells

Oguma Keisuke, Oshima Hiroko, Aoki Masahiro, Uchio Ryusei, Naka Kazuhito, Nakamura Satoshi, Hirao Atsushi, Saya Hideyuki, Taketo Makoto Mark, Oshima Masanobu

EMBO Journal 27 ( 12 ) 1671 - 1681 2008.06

ISSN 0261-4189

All Rights Reserved.The activation of Wnt/β-catenin signalling has an important function in gastrointestinal tumorigenesis. It has been suggested that the promotion of Wnt/β-catenin activity beyond the threshold is important for carcinogenesis. We herein investigated the role of macrophages in the promotion of Wnt/β-catenin activity in gastric tumorigenesis. We found β-catenin nuclear accumulation in macrophage-infiltrated dysplastic mucosa of the K19-Wnt1 mouse stomach. Moreover, macrophage depletion in Apc ^Δ716 mice resulted in the suppression of intestinal tumorigenesis. These results suggested the role of macrophages in the activation of Wnt/β-catenin signalling, which thus leads to tumour development. Importantly, the conditioned medium of activated macrophages promoted Wnt/β-catenin signalling in gastric cancer cells, which was suppressed by the inhibition of tumour necrosis factor (TNF)-α. Furthermore, treatment with TNF-α induced glycogen synthase kinase 3β (GSK3β) phosphorylation, which resulted in the stabilization of β-catenin. We also found that Helicobacter infection in the K19-Wnt1 mouse stomach caused mucosal macrophage infiltration and nuclear β-catenin accumulation. These results suggest that macrophage-derived TNF-α promotes Wnt/β-catenin signalling through inhibition of GSK3β, which may contribute to tumour development in the gastric mucosa.

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HBV X protein targets hBubR1, which induces dysregulation of the mitotic checkpoint

Kim S., Park S. Y., Yong H., Famulski J. K., Chae S., Lee J. H., Kang C. M., Saya H., Chan G. K., Cho H.

Oncogene 27 ( 24 ) 3457 - 3464 2008.05

ISSN 0950-9232

Accurate chromosomal segregation is monitored by the mitotic checkpoint, and an increased rate of chromosomal missegregation leads to chromosomal instability (CIN). Here, we demonstrate that the HBV X protein (HBx) binds BubR1, a component of the mitotic checkpoint complex and co-localizes with BubR1 at the kinetochores. HBx binding to BubR1 attenuates the association between BubR1 and CDC20, an activator of the anaphase-promoting complex/cyclosome (APC/C) and induces slippage of mitotic arrest in the presence of microtubule poisons. In addition, HBx binding to BubR1 results in the accumulation of lagging chromosomes and chromosome bridges. In contrast, a C-terminally truncated HBx mutant (HBx^1-100) fails to bind BubR1 and does not cause aberrant chromosomal segregation. This provides a novel mechanism for dysregulation of the mitotic checkpoint by a viral pathogen linking it to the accumulation of chromosomal instability in HBV-associated hepatocarcinogenesis.

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EGFR and ErbB2 are functionally coupled to CD44 and regulate shedding, internalization and motogenic effect of CD44

Pályi-Krekk Zsuzsanna, Barok Márk, Kovács Tamás, Saya Hideyuki, Nagano Osamu, Szöllosi János, Nagy Peter

Cancer Letters 263 ( 2 ) 231 - 242 2008.05

ISSN 0304-3835

Activation of the ErbB family of receptor tyrosine kinases is involved in a range of human cancers. Transmembrane signaling mediated by ErbB proteins is stimulated by peptide growth factors and is blocked by monoclonal antibodies such as trastuzumab and pertuzumab. ErbB receptors exert their function in conjunction with non-ErbB proteins, e.g. CD44. Here we show that epidermal growth factor (EGF) and heregulin induce CD44 shedding in JIMT-1, an ErbB2-overexpressing cell line resistant to trastuzumab, accompanied by internalization and intramembrane proteolysis of CD44 and enhanced cellular motility. These effects of EGF and heregulin are blocked by pertuzumab. Trastuzumab inhibits the heregulin- and hyaluronan oligosaccharide-induced shedding and internalization of CD44 and their motogenic effect. Trastuzumab also blocks CD44 shedding from JIMT-1 xenograft tumors in vivo. At the same time the internalization rate of trastuzumab is increased by hyaluronan oligosaccharide treatment in vitro. Our experiments point to an unexpected, but potentially important mechanism of action of ErbB receptor-targeted monoclonal antibodies used in the treatment of cancer.

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Neurofibromatosis type 1 (NF1) tumor suppressor, neurofibromin, regulates the neuronal differentiation of PC12 cells via its associating protein, CRMP-2

Patrakitkomjorn Siriporn, Kobayashi Daiki, Morikawa Takashi, Wilson Masayo Morifuji, Tsubota Nobuyuki, Irie Atsushi, Ozawa Tatsuya, Aoki Masashi, Arimura Nariko, Kaibuchi Kozo, Saya Hideyuki, Araki Norie

Journal of Biological Chemistry 283 ( 14 ) 9399 - 9413 2008.04

ISSN 0021-9258

Neurofibromatosis type 1 (NF1) tumor suppressor gene product, neurofibromin, functions in part as a Ras-GAP, a negative regulator of Ras. Neurofibromin is implicated in the neuronal abnormality of NF1 patients; however, the precise cellular function of neurofibromin has yet to be clarified. Using proteomic strategies, we identified a set of neurofibromin-associating cellular proteins, including axon regulator CRMP-2 (Collapsin response mediator protein-2). CRMP-2 directly bound to the C-terminal domain of neurofibromin, and this association was regulated by the manner of CRMP-2 phosphorylation. In nerve growth factor-stimulated PC12 cells, neurofibromin and CRMP-2 co-localized particularly on the distal tips and branches of extended neurites. Suppression of neurofibromin using NF1 small interfering RNA significantly inhibited this neurite outgrowth and up-regulated a series of CRMP-2 phosphorylations by kinases identified as CDK5, GSK-3b, and Rho kinase. Overexpression of the NF1-RAS-GAP-related domain rescued these NF1 small interfering RNA-induced events. Our results suggest that neurofibromin regulates neuronal differentiation by performing one or more complementary roles. First, neurofibromin directly regulates CRMP-2 phosphorylation accessibility through the complex formation. Also, neurofibromin appears to indirectly regulate CRMP-2 activity by suppressing CRMP-2-phosphorylating kinase cascades via its Ras-GAP function. Our study demonstrates that the functional association of neurofibromin and CRMP-2 is essential for neuronal cell differentiation and that lack of expression or abnormal regulation of neurofibromin can result in impaired function of neuronal cells, which is likely a factor in NF1-related pathogenesis.

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Visualizing the dynamics of p21^Waf1/Cip1 cyclin-dependent kinase inhibitor expression in living animals

Ohtani Naoko, Imamura Yuko, Yamakoshi Kimi, Hirota Fumiko, Nakayama Rika, Kubo Yoshiaki, Ishimaru Naozumi, Takahashi Akiko, Hirao Atsushi, Shimizu Takatsune, Mann David J., Saya Hideyuki, Hayashi Yoshio, Arase Seiji, Matsumoto Mitsuru, Nakao Kazuki, Hara Eiji

Proceedings of the National Academy of Sciences of the United States of America 104 ( 38 ) 15034 - 15039 2007.09

ISSN 0027-8424

Although the role of p21^Waf1/Cip1 gene expression is well documented in various cell culture studies, its in vivo roles are poorly understood. To gain further insight into the role of p21^Waf1/Cip1 gene expression in vivo, we attempted to visualize the dynamics of p21 ^Waf1/Cip1 gene expression in living animals. In this study, we established a transgenic mice line (p21-p-luc) expressing the firefly luciferase under the control of the p21^Waf1/Cip1 gene promoter. In conjunction with a noninvasive bioluminescent imaging technique, p21-p-luc mice enabled us to monitor the endogenous p21^Waf1/Cip1 gene expression in vivo. By monitoring and quantifying the p21^Waf1/Cip1 gene expression repeatedly in the same mouse throughout its entire lifespan, we were able to unveil the dynamics of p21^Waf1/Cip1 gene expression in the aging process. We also applied this system to chemically induced skin carcinogenesis and found that the levels of p21^Waf1/Cip1 gene expression rise dramatically in benign skin papillomas, suggesting that p21^Waf1/Cip1 plays a preventative role(s) in skin tumor formation. Surprisingly, moreover, we found that the level of p21^Waf1/Cip1 expression strikingly increased in the hair bulb and oscillated with a 3-week period correlating with hair follicle cycle progression. Notably, this was accompanied by the expression of p63 but not p53. This approach, together with the analysis of p21 ^Waf1/Cip1 knockout mice, has uncovered a novel role for the p21 ^Waf1/Cip1 gene in hair development. These data illustrate the unique utility of bioluminescence imaging in advancing our understanding of the timing and, hence, likely roles of specific gene expression in higher eukaryotes.

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The UHRF family

Bronner Christian, Achour Mayada, Arima Yoshimi, Chataigneau Thierry, Saya Hideyuki, Schini-Kerth Valérie B.

Pharmacology and Therapeutics 115 ( 3 ) 419 - 434 2007.09

ISSN 0163-7258

In this paper, we review the current literature about the UHRF family that in particular includes the UHRF1 and UHRF2 genes. Its members play a fundamental role in cell proliferation through different structural domains. These domains include a ubiquitin-like domain (NIRF_N), a plant homeodomain (PHD) domain, a SRA domain and a RING domain. The SRA domain has only been observed in this family probably conferring unique properties to it. The unique enzymatic activity so far identified in this family involves the RING finger that contains a ubiquitin E3 ligase activity toward, for instance, histones. The physiological roles played by the UHRF family are most likely exerted during embryogenic development and when proliferation is required in adults. Interestingly, UHRF members are putative oncogenes regulated by tumor suppressor genes, but they exert also a feedback control on these latter. Finally, we propose some new roles for this family, including regulation and/or inheritance of the epigenetic code. Alteration of these regulatory mechanisms, such as those occurring in cancer cells, may be involved in carcinogenesis. The reasons why the UHRF family could be an interesting target for developing anticancer drugs is also developed.

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Differential levels of human leukocyte antigen-class I, multidrug-resistance 1 and androgen receptor expressions in untreated prostate cancer cells

Homma Shigenori, Komohara Yoshihiro, Harada Mamoru, Saya Hideyuki, Todo Satoru, Itoh Kyogo, Noguchi Masanori

Oncology Reports 18 ( 2 ) 343 - 346 2007.08

ISSN 1021-335X

Tumors are highly robust and maintain their proliferative potential against a wide range of both host-defense mechanisms and anticancer therapies. One of the approaches to overcome cancer robustness could be combined therapy in which each modality imposes independent selective pressures against the acquired mutation of cancer. To develop such a therapy, it is crucial to understand the magnitude of acquired mutations. In this study, we investigated the levels of human leukocyte antigen (HLA)-class I, multidrug-resistance 1 (MDR1), and androgen receptor (AR) expressions in untreated prostate cancers harvested by radical prostatectomy. The mean percentages of cancer cells expressing HLA-class I, MDR and AR among the 10 cancer samples were 41, 35 and 74%, respectively. In addition, double-staining of HLA and MDR revealed the four definite populations (HLA⁺/MDR⁺, HLA+/MDR^-, HLA-/MDR⁺and HLA^-/MDR^-) in cancer tissues from the majority of cancer patients tested, and the mean percentages of cells expressing these combinations were 13, 29, 22 and 38%, respectively. Similar results were obtained by double-staining of HLA and AR, except for 2 cases in which HLA ^-/AR⁺cancer cells predominated. These results indicated that untreated prostate cancer cells acquired a wide range of genomic mutations, which may have been caused by internal host pressure to eliminate malignant cells, and would provide evidence of the robustness of untreated prostate cancer.

Zoledronate-induced S phase arrest and apoptosis accompanied by DNA damage and activation of the ATM/Chk1/cdc25 pathway in human osteosarcoma cells

Iguchi Toyotaka, Miyakawa Yoshitaka, Saito Kaori, Nakabayashi Chika, Nakanishi Makoto, Saya Hideyuki, Ikeda Yasuo, Kizaki Masahiro

International Journal of Oncology 31 ( 2 ) 285 - 291 2007.08

ISSN 1019-6439

Osteosarcoma is one of the most common primary malignant tumors of the bone in children and adolescents. Some patients continue to have a poor prognosis, as they have metastatic disease and frequent occurrence of drug resistance. Zoledronate is a nitrogen-containing bisphosphonate that has been used for the treatment of hypercalcemia and bone metastasis, because it induces apoptosis in osteoclasts and tumor cells by inhibiting the isoprenylation of intracellular small G proteins. Besides inhibiting isoprenylation, little is known about the manner by which bisphosphonates inhibit cellular proliferation and induce apoptosis. This prompted us to investigate the inhibitory effects of zoledronate in human osteosarcoma cell lines, HOS and MG63. HOS cells accumulated in S phase around 6 h after treatment with 10 μM zoledronate, followed by apoptosis. When HOS cells were treated with zoledronate, ATM kinase and its substrate, check-point kinase (Chk)1, were phosphorylated. Zoledronate also induced phosphorylation of cdc25a (Thr506) in HOS cells, which is a substrate of Chk1, and its phosphorylation is known to be critical for S phase arrest. Following treatment with zoledronate, phosphorylated histone H2AX (γ-H2AX) displayed patterns of nuclear foci in HOS cells. As γ-H2AX accumulates at dsDNA breaks, these results demonstrate that zoledronate induced DNA damage and S phase arrest, accompanied by activation of the ATM/Chk1/cdc25 pathway in a human osteosarcoma cell line.

Serine protease Omi/HtrA2 targets WARTS kinase to control cell proliferation

Kuninaka S., Iida S. I., Hara T., Nomura M., Naoe H., Morisaki T., Nitta M., Arima Y., Mimori T., Yonehara S., Saya H.

Oncogene 26 ( 17 ) 2395 - 2406 2007.04

ISSN 0950-9232

The serine protease Omi/HtrA2 was initially regarded as a proapoptotic molecule that proteolyses several proteins to induce cell death. Recent studies, however, indicate that loss of Omi protease activity increases susceptibility to stress-induced cell death. These complicated findings suggest that the protease activity of Omi is involved not only in apoptosis but also in cellular homeostasis. However, the targets which Omi uses to mediate this novel process are unknown. Previously, we showed that WARTS (WTS)/large tumor-suppressor 1 mitotic kinase interacts with the protein/discs-large protein/zonula (PDZ) domain of Omi and promotes its protease activity. We now report that WTS is a substrate for Omi protease activity, thus it is not only a regulator but also a downstream target of this protease. Interaction with Omi PDZ domain is required for WTS to be proteolysed. When caspase-9-deficient mouse embryonic fibroblasts (MEFs) were treated with staurosporine, WTS was proteolysed by activated endogenous Omi without induction of cell death. Therefore, protease activity of Omi and proteolysis of WTS are not necessarily required for cell death. We found that depletion of Omi from HeLa cells results in accelerated cell proliferation despite no significant change in the duration of mitosis. The depletion of WTS showed the same effect on S phase progression. Therefore, WTS proteolytic fragment(s) generated by Omi may act as an inhibitor of G1/S progression. Our data reveal a role for Omi-mediated processing of WTS in negative regulation of cell cycle progression at interphase, suggesting a novel function of Omi other than apoptosis.

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ADAM-17 associated with CD44 cleavage and metastasis in oral squamous cell carcinoma

Takamune Yasuo, Ikebe Tetsuro, Nagano Osamu, Nakayama Hideki, Ota Kazutoshi, Obayashi Takehisa, Saya Hideyuki, Shinohara Masanori

Virchows Archiv 450 ( 2 ) 169 - 177 2007.02

ISSN 0945-6317

ADAM-17 (a disintegrin and metalloproteinase 17) is a membrane-anchored protein, which can cleave the ectodomain in a variety of transmembrane proteins. In the in vitro experiments with tumor cells, ADAM-17 is reported to cleave CD44, an adhesion molecule that interacts with hyaluronic acid, to promote tumor cell migration. In the present study, we examined ADAM-17 expression and CD44 cleavage in specimens from 50 patients diagnosed to have oral squamous cell carcinoma (SCC). Each specimen was divided into two pieces, one was studied by immunohistochemistry and the other was subjected to a Western blot. By coordinating the results of both analyses, ADAM-17 expression was evaluated to be high in 23 cases (46%). When CD44 cleavage was also studied by immunohistochemical staining as well as with Western blotting, CD44 cleavage was judged to be positive in 29 cases (58%). When the ADAM-17 expression level was compared with the CD44 cleavage state, most of the cases expressing high levels of ADAM-17 (87%) showed positive CD44 cleavage. The level of ADAM-17 expression was significantly correlated to the nodal metastasis and local recurrence in oral SCC. Our findings suggest that ADAM-17 is involved in CD44 cleavage and contributes to tumor progression in oral SCC.

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NDEL1 phosphorylation by Aurora-A kinase is essential for centrosomal maturation, separation, and TACC3 recruitment

Mori Daisuke, Yano Yoshihisa, Toyo-Oka Kazuhito, Yoshida Noriyuki, Yamada Masami, Muramatsu Masami, Zhang Dongwei, Saya Hideyuki, Toyoshima Yoko Y., Kinoshita Kazuhisa, Wynshaw-Boris Anthony, Hirotsune Shinji

Molecular and Cellular Biology 27 ( 1 ) 352 - 367 2007.01

ISSN 0270-7306

NDEL1 is a binding partner of LIS1 that participates in the regulation of cytoplasmic dynein function and microtubule organization during mitotic cell division and neuronal migration. NDEL1 preferentially localizes to the centrosome and is a likely target for cell cycle-activated kinases, including CDK1. In particular, NDEL1 phosphorylation by CDK1 facilitates katanin p60 recruitment to the centrosome and triggers microtubule remodeling. Here, we show that Aurora-A phosphorylates NDEL1 at Ser251 at the beginning of mitotic entry. Interestingly, NDEL1 phosphorylated by Aurora-A was rapidly downregulated thereafter by ubiquitination-mediated protein degradation. In addition, NDEL1 is required for centrosome targeting of TACC3 through the interaction with TACC3. The expression of Aurora-A phosphorylation-mimetic mutants of NDEL1 efficiently rescued the defects of centrosomal maturation and separation which are characteristic of Aurora-A-depleted cells. Our findings suggest that Aurora-A-mediated phosphorylation of NDEL1 is essential for centrosomal separation and centrosomal maturation and for mitotic entry.

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Expression of WT1 protein and correlation with cellular proliferation in glial tumors

Saya Hideyuki

Neurologia Medico-Chirurgica 47 ( 4 ) 170 2007

ISSN 0470-8105

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Aurora kinases

Saya Hideyuki

Seikagaku 79 ( 2 ) 131 - 139 2007

ISSN 0037-1017

Mitogenic signalling and the p16^INK4a-Rb pathway cooperate to enforce irreversible cellular senescence

Takahashi Akiko, Ohtani Naoko, Yamakoshi Kimi, Iida Shin Ichi, Tahara Hidetoshi, Nakayama Keiko, Nakayama Keiichi I., Ide Toshinori, Saya Hideyuki, Hara Eiji

Nature Cell Biology 8 ( 11 ) 1291 - 1297 2006.11

ISSN 1465-7392

The p16^INK4a cyclin-dependent kinase inhibitor has a key role in establishing stable G1 cell-cycle arrest through activating the retinoblastoma (Rb) tumour suppressor protein pRb^1-5 in cellular senescence. Here, we show that the p16^INK4a/ Rb-pathway also cooperates with mitogenic signals to induce elevated intracellular levels of reactive oxygen species (ROS), thereby activating protein kinase Cδ (PKCδ) in human senescent cells. Importantly, once activated by ROS, PKCδ promotes further generation of ROS, thus establishing a positive feedback loop to sustain ROS-PKCδ signalling^6-8. Sustained activation of ROS-PKCδ signalling irreversibly blocks cytokinesis, at least partly through reducing the level of WARTS (also known as LATS1), a mitotic exit network (MEN) kinase required for cytokinesis^9-11, in human senescent cells. This irreversible cytokinetic block is likely to act as a second barrier to cellular immortalization ensuring stable cell-cycle arrest in human senescent cells. These results uncover an unexpected role for the p16^INK4a-Rb pathway and provide a new insight into how senescent cell-cycle arrest is enforced in human cells.

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Molecular mechanisms of anti-cancer therapies

Saya Hideyuki

Japanese Journal of Neurosurgery 15 ( 10 ) 688 - 693 2006.10

ISSN 0917-950X

Cell cycle checkpoints block the transition from one phase of the cell cycle to the next phase until all processes are completed. Defects in these checkpoint functions lead to genomic instability, which results in the development of malignant tumors. However, it has become known that a loss of checkpoint function is directly related to sensitivity to antineoplastic treatments. Most cancer cells are deficient in G1 checkpoint function and therefore fail to arrest in the G1 phase on exposure to genotoxic agents. Instead, they accumulate temporarily in the G2 phase. However, given that the G2 checkpoint is also partially impaired in cancer cells, they are unable to maintain G2 arrest and eventually die as they enter mitosis. This process is known as mitotic catastrophe or mitotic death. Mitotic catastrophe is an important mechanism for the induction of cell death in cancer cells by antineoplastic agents. Cells entering mitosis with DNA damage arrest at metaphase for more than 10hr without chromosome segregation and subsequently die directly from metaphase. This long-term metaphase arrest before catastrophic death is termed the "precatastrophic phase". We found that spindle check is activated during the precatastrophic phase. Therefore, inhibition of the spindle checkpoint function as a result of depletion of BubR1 or Mad2 by RNAi caused cells to escape from mitotic catastrophe and led to subsequent abnormal mitosis when DNA damage is induced. Our data suggest that a dysfunction of the mitotic checkpoint in cancer cells may confer resistance to DNA-damaging agents and malignant progression. In this review article, I would like to summarize how cancer cells are terminated by DNA-damaging agents and anti-mitotic drugs. Based on these findings, I would like to propose the novel mechanisms by which tumors become insensitive to those chemotherapeutic agents.

Centrosome amplification in adult T-cell leukemia and human T-cell leukemia virus type 1 Tax-induced human T cells

Nitta Takayuki, Kanai Masayuki, Sugihara Eiji, Tanaka Masakazu, Sun Binilian, Nagasawa Toshiro, Sonoda Shunro, Saya Hideyuki, Miwa Masanao

Cancer Science 97 ( 9 ) 836 - 841 2006.09

ISSN 1347-9032

Centrosomes play pivotal roles in cell polarity, regulation of the cell cycle and chromosomal segregation. Centrosome amplification was recently described as a possible cause of aneuploidy in certain solid tumors and leukemias. ATL is a T-cell malignancy caused by HTLV-1. Although the precise mechanism of cell transformation is unclear, the HTLV-1-encoded protein, Tax, is thought to play a crucial role in leukemogenesis. Here we demonstrate that lymphocytes isolated from patients with ATL show centrosome amplification and that a human T cell line shows centrosome amplification after induction of Tax, which was suppressed by CDK inhibitors. Micronuclei formation was also observed after centrosome amplification in Tax-induced human T cells. These findings suggest that Tax deregulates CDK activity and induces centrosome amplification, which might be associated with cellular transformation by HTLV-1 and chromosomal instability in HTLV-1-infected human T cells.

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Dynamics of mitotic spindle

Kinoshita Kazuhisa, Saya Hideyuki

Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme. 51 ( 6 Suppl ) 761 - 766 2006.05

ISSN 0039-9450

Suppression of centrosome amplification after DNA damage depends on p27 accumulation

Sugihara Eiji, Kanai Masayuki, Saito Soichiro, Nitta Takayuki, Toyoshima Hideo, Nakayama Keiko, Nakayama Keiichi I., Fukasawa Kenji, Schwab Manfred, Saya Hideyuki, Miwa Masanao

Cancer Research 66 ( 8 ) 4020 - 4029 2006.04

ISSN 0008-5472

The centrosome plays a fundamental role in cell division, cell polarity, and cell cycle progression. Centrosome duplication is mainly controlled by cyclin-dependent kinase 2 (CDK2)/cyclin E and cyclin A complexes, which are inhibited by the CDK inhibitors p21^Cip1 and p27^Kip1. It is thought that abnormal activation of CDK2 induces centrosome amplification that is frequently observed in a wide range of aggressive tumors. We previously reported that overexpression of the oncogene MYCN leads to centrosome amplification after DNA damage in neuroblastoma cells. We here show that centrosome amplification after γ-irradiation was caused by suppression of p27 expression in MYCN-overexpressing cells. We further show that p27^-/- and p27^+/- mouse embryonic fibroblasts and p27-silenced human cells exhibited a significant increase in centrosome amplification after DNA damage. Moreover, abnormal mitotic cells with amplified centrosomes were frequently observed in p27-silenced cells. In response to DNA damage, the level of p27 gradually increased in normal cells independently of the ataxia telangiectasia mutated/p53 pathway, whereas Skp2, an F-box protein component of an SCF ubiquitin ligase complex that targets p27, was reduced. Additionally, p27 levels in MYCN-overexpressing cells were restored by treatment with Skp2 small interfering RNA, indicating that down-regulation of p27 by MYCN was due to high expression of Skp2. These results suggest that the accumulation of p27 after DNA damage is required for suppression of centrosome amplification, thereby preventing chromosomal instability.

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Antitumor effect of E1A in ovarian cancer by cytoplasmic sequestration of activated ERK by PEA15

Bartholomeusz C., Itamochi H., Nitta M., Saya H., Ginsberg M. H., Ueno N. T.

Oncogene 25 ( 1 ) 79 - 90 2006.01

ISSN 0950-9232

The adenovirus type 5 gene E1A is known to suppress tumorigenicity by transcriptionally downregulating HER-2/neu (HER2) or by inducing apoptosis. We show here that E1A also suppressed the tumorigenicity of the low-HER2-expressing ovarian cancer cell line OVCAR-3 by decreasing cell proliferation. We further found that the mechanism responsible for this reduced proliferation is the presence of PEA15 (phosphoprotein enriched in astrocytes), which is upregulated by E1A in ovarian cancer; PEA15 promotes translocation of ERK from the nucleus to the cytoplasm, leading to inhibition of ERK-dependent transcription and proliferation. Indeed, siRNA-mediated knockdown of PEA15 expression in OVCAR-3 stable E1A transfectants resulted in a nuclear accumulation of the active form of ERK, followed by an increase in Elk-1 activity, DNA synthesis, and anchorage-independent growth. Finally, PEA15 by itself suppressed colony formation in breast and ovarian cancer cell lines, in which E1A is known to have antitumor activity. We conclude that part of the antitumor effect of E1A in ovarian cancer results from cytoplasmic sequestration of the activated form of ERK by PEA15.

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Preliminary individualized chemotherapy for malignant astrocytomas based on O⁶-methylguanine-deoxyribonucleic acid methyltransferase methylation analysis

Saya Hideyuki

Neurologia Medico-Chirurgica 46 ( 8 ) 394 2006

ISSN 0470-8105

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The neurofibromatosis type 1 gene product neurofibromin enhances cell motility by regulating actin filament dynamics via the Rho-ROCK-LIMK2-cofilin pathway

Ozawa Tatsuya, Araki Norie, Yunoue Shunji, Tokuo Hiroshi, Feng Liping, Patrakitkomjorn Siriporn, Hara Toshihiro, Ichikawa Yasuko, Matsumoto Kunio, Fuji Kiyotaka, Saya Hideyuki

Journal of Biological Chemistry 280 ( 47 ) 39524 - 39533 2005.11

ISSN 0021-9258

Neurofibromin is a neurofibromatosis type 1 (NF1) tumor suppressor gene product with a domain that acts as a GTPase-activating protein and functions, in part, as a negative regulator of Ras. Loss of neurofibromin expression in NF1 patients is associated with elevated Ras activity and increased cell proliferation, predisposing to a variety of tumors of the peripheral and central nervous systems. We show here, using the small interfering RNA (siRNA) technique, that neurofibromin dynamically regulates actin cytoskeletal reorganization, followed by enhanced cell motility and gross cell aggregation in Matrigel matrix. NF1 siRNA induces characteristic morphological changes, such as excessive actin stress fiber formation, with elevated negative phosphorylation levels of cofilin, which regulates actin cytoskeletal reorganization by depolymerizing and severing actin filaments. We found that the elevated phosphorylation of cofilin in neurofibromin-depleted cells is promoted by activation of a Rho-ROCK-LIMK2 pathway, which requires Ras activation but is not transduced through three major Ras-mediated downstream pathways via Raf, phosphatidylinositol 3-kinase, and RalGEF. In addition, the exogenous expression of the NF1-GTPase-activating protein-related domain suppressed the NF1 siRNA-induced phenotypes. Neurofibromin was demonstrated to play a significant role in the machinery regulating cell proliferation and in actin cytoskeletal reorganization, which affects cell motility and adhesion. These findings may explain, in part, the mechanism of multiple neurofibroma formation in NF1 patients.

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The tumor suppressor WARTS activates the Omi/HtrA2-dependent pathway of cell death

Kuninaka Shinji, Nomura Masanobu, Hirota Toru, Iida Shin Ichi, Hara Toshihiro, Honda Shinobu, Kunitoku Naoko, Sasayama Takashi, Arima Yoshimi, Marumoto Tomotoshi, Koja Kageharu, Yonehara Shin, Saya Hideyuki

Oncogene 24 ( 34 ) 5287 - 5298 2005.08

ISSN 0950-9232

Drosophila tumor suppressor WARTS (Wts) is an evolutionally conserved serine/threonine kinase and parti cipates in a signaling complex that regulates both proliferation and apoptosis to ensure the proper size and shape of the fly. Human counterparts of this complex have been found to be frequently downregulated or mutated in cancers. WARTS, a human homolog of Wts, is also known as tumor suppressor and mitotic regulator, but its molecular implications in tumorigenesis are still obscure. Here, we show that WARTS binds via its C-terminus to the PDZ domain of a proapoptotic serine protease Omi/ HtrA2. Depletion of WARTS inhibited Omi/HtrA2-mediated cell death, whereas overexpression of WARTS promoted this process. Furthermore, WARTS can enhance the protease activity of Omi/HtrA2 both in vivo and in vitro. Activation of Omi/HtrA2-mediated cell death is thus a potential mechanism for the tumor suppressive activity of WARTS.

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Multiple roles of vertebrate REV genes in DNA repair and recombination

Okada Takashi, Sonoda Eiichiro, Yoshimura Michio, Kawano Yoshiaki, Saya Hideyuki, Kohzaki Masaoki, Takeda Shunichi

Molecular and Cellular Biology 25 ( 14 ) 6103 - 6111 2005.07

ISSN 0270-7306

In yeast, Rev1, Rev3, and Rev7 are involved in translesion synthesis over various kinds of DNA damage and spontaneous and UV-induced mutagenesis. Here, we disrupted Rev1, Rev3, and Rev7 in the chicken B-lymphocyte line DT40. REV1 ^-/- REV3^-/- REV7^-/- cells showed spontaneous cell death, chromosomal instability/fragility, and hypersensitivity to various genotoxic treatments as observed in each of the single mutants. Surprisingly, the triple-knockout cells showed a suppressed level of sister chromatid exchanges (SCEs), which may reflect postreplication repair events mediated by homologous recombination, while each single mutant showed an elevated SCE level. Furthermore, REV1^-/- cells as well as triple mutants showed a decreased level of immunoglobulin gene conversion, suggesting participation of Rev1 in a recombination-based pathway. The present study gives us a new insight into cooperative function of three Rev molecules and the Polζ (Rev3-Rev7)-independent role of Rev1 in vertebrate cells.

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Over-expression of Aurora-A targets cytoplasmic polyadenylation element binding protein and promotes mRNA polyadenylation of Cdk1 and cyclin B1

Sasayama Takashi, Marumoto Tomotoshi, Kunitoku Naoko, Zhang Dongwei, Tamaki Norihiko, Kohmura Eiji, Saya Hideyuki, Hirota Toru

Genes to Cells 10 ( 7 ) 627 - 638 2005.07

ISSN 1356-9597

Aurora-A is a centrosomal serine-threonine kinase that regulates mitosis. Over-expression of Aurora-A has been found in a wide range of tumors and has been implicated in oncogenic transformation. However, how Aurora-A over-expression contributes to promotion of carcinogenesis remains elusive. Immunohistochemical analysis of breast tumors revealed that over-expressed Aurora-A is not restricted to the centrosomes but is also found in the cytoplasm. This over-expressed Aurora-A appeared to be phosphorylated on Thr288, which is known to be required for its enzymatic activation. In analogy to Aurora-A's role in oocyte maturation and the early embryonic cell cycle, here we investigated whether ectopically over-expressed Aurora-A can similarly stimulate polyadenylation of mRNA in human somatic cultured cells by interacting with a human ortholog of cytoplasmic polyadenylation element binding protein, h-CPEB. In vitro experiments revealed that Aurora-A binds directly to, and phosphorylates, h-CPEB.We found that polyadenylation of mRNA tails of cyclin B1 and Cdk1 was synergistically stimulated when Aurora-A and h-CPEB were overexpressed, and they were further promoted in the presence of an Aurora-A activator Ajuba. Our results suggest a function of ectopically over-expressed Aurora-A that might be relevant for carcinogenesis.

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Molecular mechanism of tumorigenesis by cell cycle regulation and new therapeutical approaches

Saya Hideyuki

Japanese Journal of Neurosurgery 14 ( 6 ) 373 - 378 2005.06

ISSN 0917-950X

Cell cycle checkpoints prevent transition from one phase of the cell cycle to the next until all processes of the present phase are completed. Defects in the checkpoint functions result in gene mutations and chromosome damages, which contribute to the development and progression of tumors. However, loss of checkpoint function in some cancer cells is considered to be associated with their sensitivity to antineoplastic treatments such as chemotherapy and radiation therapy. Most cancer therapies target cell cycle checkpoints by activating checkpoint-mediated cell death or by enhancing chemical sensitivity due to loss of checkpoint function. By treatment with genotoxic agents, cancer cells, which generally have impairment of checkpoint functions, initially arrest in the G2 phase of the cell cycle but are unable to maintain cell-cycle arrest. Those cells eventually die as they entered mitosis. This process is called 'mitotic catastrophe'. This review discusses the critical relationship between mitotic checkpoint function and sensitivity of cancer cells to anti-tumor therapies.

Transcriptional blockade induces p53-dependent apoptosis associated with translocation of p53 to mitochondria

Arima Yoshimi, Nitta Masayuki, Kuninaka Shinji, Zhang Dongwei, Fujiwara Toshiyoshi, Taya Yoichi, Nakao Mitsuyoshi, Saya Hideyuki

Journal of Biological Chemistry 280 ( 19 ) 19166 - 19176 2005.05

ISSN 0021-9258

The tumor suppressor p53 functions as a transcriptional activator to induce cell cycle arrest and apoptosis in response to BNA damage. Although p53 was also shown to mediate apoptosis in a manner independent of its transactivation activity, the mechanism and conditions that trigger such cell death have remained largely unknown. We have now shown that inhibition of RNA polymerase II-mediated transcription by α-amanitin or RNA interference induced p53-dependent apoptosis. Inhibition of pol II-mediated transcription resulted in down-regulation of p21^Cip1, which was caused by both transcriptional suppression and protein degradation, despite eliciting p53 accumulation, allowing the cells to progress into S phase and then to undergo apoptosis. This cell death did not require the transcription of p53 target genes and was preceded by translocation of the accumulated p53 to mitochondria. Our data thus suggested that blockade of pol II-mediated transcription induced p53 accumulation in mitochondria and was the critical factor for eliciting p53-dependent but transcription-independent apoptosis.

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RNA interference targeting aurora kinase A suppresses tumor growth and enhances the taxane chemosensitivity in human pancreatic cancer cells

Hata Tatsuo, Furukawa Toru, Sunamura Makoto, Egawa Shinichi, Motoi Fuyuhiko, Ohmura Noriyuki, Marumoto Tomotoshi, Saya Hideyuki, Horii Akira

Cancer Research 65 ( 7 ) 2899 - 2905 2005.04

ISSN 0008-5472

AURKA/STK15/BTAK, the gene encoding Aurora A kinase that is involved in the regulation of centrosomes and segregation of chromosomes, is frequently amplified and overexpressed in various kinds of human cancers, including pancreatic cancer. To address its possibility as a therapeutic target for pancreatic cancer, we employed the RNA interference technique to knockdown AURKA expression and analyzed its phenotypes. We found that the specific knockdown of AURKA in cultured pancreatic cancer cells strongly suppressed in vitro cell growth and in vivo tumorigenicity. The knockdown induced the accumulation of cells in the G₂-M phase and eventual apoptosis. Furthermore, we observed a synergistic enhancement of the cytotoxicity of taxanes, a group of chemotherapeutic agents impairing G₂-M transition, by the RNA interference-mediated knockdown of AURKA. These results indicate that inhibition of AURKA expression can result in potent antitumor activity and chemosensitizing activity to taxanes in human pancreatic cancer.

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Chfr is required for tumor suppression and Aurora A regulation

Yu Xiaochun, Minter-Bykhouse Katherine, Malureanu Liviu, Zhao Wei Meng, Zhang Dongwei, Merkle Carolin J., Ward Irene M., Saya Hideyuki, Fang Guowei, Van Deursen Jan, Chen Junjie

Nature Genetics 37 ( 4 ) 401 - 406 2005.04

ISSN 1061-4036

Tumorigenesis is a consequence of loss of tumor suppressors and activation of oncogenes. Expression of the mitotic checkpoint protein Chfr is lost in 20-50% of primary tumors and tumor cell lines. To explore whether downregulation of Chfr contributes directly to tumorigenesis, we generated Chfr knockout mice. Chfr-deficient mice are cancer-prone, develop spontaneous tumors and have increased skin tumor incidence after treatment with dimethylbenz(a)anthracene. Chfr deficiency leads to chromosomal instability in embryonic fibroblasts and regulates the mitotic kinase Aurora A, which is frequently upregulated in a variety of tumors. Chfr physically interacts with Aurora A and ubiquitinates Aurora A both in vitro and in vivo. Collectively, our data suggest that Chfr is a tumor suppressor and ensures chromosomal stability by controlling the expression levels of key mitotic proteins such as Aurora A.

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Aurora-A - A guardian of poles

Marumoto Tomotoshi, Zhang Dongwei, Saya Hideyuki

Nature Reviews Cancer 5 ( 1 ) 42 - 50 2005.01

ISSN 1474-175X

The three human homologues of Aurora kinases (A, B and C) are essential for proper execution of various mitotic events and are important for maintaining genomic integrity. Aurora-A is mainly localized at spindle poles and the mitotic spindle during mitosis, where it regulates the functions of centrosomes, spindles and kinetochores required for proper mitotic progression. Recent studies have revealed that Aurora-A is frequently overexpressed in various cancer cells, indicating its involvement in tumorigenesis. What are the normal physiological roles of Aurora-A, how are these regulated and how might the enzyme function during tumorigenesis?

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Molecular mechanisms of brain tumor invasion

Saya Hideyuki

Nippon rinsho. Japanese journal of clinical medicine 63 Suppl 9 61 - 67 2005

ISSN 0047-1852

Cell Death

Saya Hideyuki

Advances in Bioethics 8 209 - 223 2005

ISSN 1479-3709

Natural selection is the mechanism of evolution originally proposed by Charles Darwin. This process is driven by mutation and death. Therefore, "individual death" is a critical event for evolution of species. Analogous to the significance of individual death in species evolution, recent biological observations have revealed that "cell death" is an important event for maintenance of individuals and offspring. Cell death is caused not only by injuries and pathological conditions, but also by programed intracellular signals. This type of programed cell death is called "apoptosis." Apoptosis signals are regulated in mitochondria, eukaryotic organelles of symbiotic origin, and play a critical role in survival of individuals by eliminating some cells.

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Observation of the intracellular behavior of recombinant Yersinia pseudotuberculosis invasin protein

Koga Hisashi, Izawa Ichiro, Araki Norie, Saya Hideyuki, Mimori Tatsuyuki

Microbiology and Immunology 49 ( 4 ) 297 - 302 2005

ISSN 0385-5600

In this study, we observed the intracellular behavior of recombinant invasin, a 103-kDa outer membrane protein of Yersinia pseudotuberculosis. To mimic the in vivo behavior of bacterial invasin, a polyvalent form of invasin was generated by incubation of biotinylated GST-fused invasin C-terminal portion protein (GST-INVS) with avidin. Several experiments confirmed that the recombinant invasin could consistently reproduce the invasin-mediated entry to mammalian epithelial cells. We analyzed the molecular kinetics of polyvalent INVS by western blotting, ¹²⁵I-uptake, and immunofluorescent microscopy. The internalized polyvalent INVS was rapidly translocated to the RIPA-insoluble (polymerized-actin enriched) fraction and formed cytoplasmic vesicles, while monovalent invasin did not show such kinetics. From these observations, we concluded that our bacterial-free system is able to analyze the action of invasin for Yersinia pseudotuberculosis entry.

Overexpression of heparin-binding growth-associated molecule in malignant glioma cells

Ducati Alessandro, Mut Melike, Shaffrey Mark E., Laws Edward R., Saya Hideyuki

Neurologia Medico-Chirurgica 44 ( 12 ) 644 - 645 2004.12

ISSN 0470-8105

Mechanism and biological significance of CD44 cleavage

Nagano Osamu, Saya Hideyuki

Cancer Science 95 ( 12 ) 930 - 935 2004.12

ISSN 1347-9032

There are multiple steps in the metastasis of cancer cells. Tumor cells must first detach from the tumor mass and invade the surrounding extracellular matrix (ECM). In this step, cell surface adhesion molecules play an important role in the interaction between the cells and their microenvironments. CD44 is an adhesion molecule that interacts with hyaluronic acid (HA) and is implicated in a wide variety of physiological and pathological processes. Recently, proteolytic cleavages of CD44 have been emerging as key regulatory events for the CD44 dependent cell-matrix interaction and signaling pathway. CD44 undergoes sequential proteolytic cleavages in the ectodomain and intramembranous domain, resulting in the release of a CD44 intracellular domain (ICD) fragment. The ectodomain cleavage of CD44 is triggered by multiple stimulations and contributes to the regulation of cell attachment to and migration on HA matrix. The ectodomain cleavage subsequently induces the intramembranous cleavage, which is mediated by presenilin (PS)-dependent γ-secretase. The intramembranous cleavage generates CD44ICD, which acts as a signal transduction molecule; it is translocated to the nucleus and activates transcription. An understanding of the underlying mechanism of these cleavages of CD44 could provide novel therapeutic targets for cancer cell invasion and metastasis.

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Cre-loxP-controlled periodic Aurora-A overexpression induces mitotic abnormalities and hyperplasia in mammary glands of mouse models

Zhang Dongwei, Hirota Toru, Marumoto Tomotoshi, Shimizu Michio, Kunitoku Naoko, Sasayama Takashi, Arima Yoshimi, Feng Liping, Suzuki Misao, Takeya Motohiro, Saya Hideyuki

Oncogene 23 ( 54 ) 8720 - 8730 2004.11

ISSN 0950-9232

Aurora-A, a serine/threonine mitotic kinase, was reported to be overexpressed in various human cancers, and its overexpression induces aneuploidy, centrosome amplification and tumorigenic transformation in cultured human and rodent cells. However, the underlying mechanisms and pathological settings by which Aurora-A promotes tumorigenesis are largely unknown. Here, we created a transgenic mouse model to investigate the involvement of Aurora-A overexpression in the development of mammary glands and tumorigenesis using a Cre-loxP system. The conditional expression of Aurora-A resulted in significantly increased binucleated cell formation and apopiosis in the mammary epithelium. The surviving mammary epithelial cells composed hyperplastic areas after a short latency. Induction of Aurora-A overexpression in mouse embryonic fibroblasts prepared from the transgenic mice also led to aberrant mitosis and binucleated cell formation followed by apoptosis. The levels of p53 protein were remarkably increased in these Aurora-A-overexpressing cells, and the apoptosis was significantly suppressed by deletion of p53. Given that no malignant tumor formation was found in the Aurora-A-overexpressing mouse model after a long latency, additional factors, such as p53 inactivation, are required for the tumorigenesis of Aurora-A-overexpressing mammary epithelium. Our findings indicated that this mouse model is a useful system to study the physiological roles of Aurora-A and the genetic pathways of Aurora-A-induced carcinogenesis.

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Mechanism and biological significance of sequential cleavages of CD44 - A potential molecular target for cancer therapy

Nagano Osamu, Murakami Daizo, Saya Hideyuki

Biotherapy 18 ( 5 ) 405 - 411 2004.09

ISSN 0914-2223

Spindle checkpoint function is required for mitotic catastrophe induced by DNA-damaging agents

Nitta Masayuki, Kobayashi Osamu, Honda Shinobu, Hirota Toru, Kuninaka Shinji, Marumoto Tomotoshi, Ushio Yukitaka, Saya Hideyuki

Oncogene 23 ( 39 ) 6548 - 6558 2004.08

ISSN 0950-9232

Mitotic catastrophe is an important mechanism for the induction of cell death in cancer cells by antineoplastic agents that damage DNA. This process is facilitated by defects in the G₁ and G₂ checkpoints of the cell cycle that are apparent in most cancer cells and which allow the cells to enter mitosis with DNA damage. We have now characterized the dynamics of mitotic catastrophe induced by DNA-damaging agents in p53-deficient cancer cells. Cells that entered mitosis with DNA damage transiently arrested at metaphase for more than 10h without segregation of chromosomes and subsequently died directly from metaphase. In those metaphase arrested precatastrophic cells, anaphase-promoting complex appeared to be inactivated and BubR1 was persistently localized at kinetochores, suggesting that spindle checkpoint is activated after the DNA damage. Furthermore, suppression of spindle checkpoint fonction by BnbR1 or Mad2 RNA interference in the DNA damaged cells led to escape from catastrophic death and to subsequent abnormal mitosis. Dysfunction of the spindle checkpoint in p53-deficient cancer cells is thus likely a critical factor in resistance to DNA-damaging therapeutic agents.

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Tumor suppressor WARTS ensures genomic integrity by regulating both mitotic progression and G₁ tetraploidy checkpoint function

Iida Shin Ichi, Hirota Toru, Morisaki Tetsuro, Marumoto Tomotoshi, Hara Toshihiro, Kuninaka Shinji, Honda Shinobu, Kosai Ken Ichiro, Kawasuji Michio, Pallas David C., Saya Hideyuki

Oncogene 23 ( 31 ) 5266 - 5274 2004.07

ISSN 0950-9232

Defects in chromosomes or mitotic spindles activate the spindle checkpoint, resulting in cell cycle arrest at prometaphase. The prolonged activation of spindle checkpoint generally leads to mitotic exit without segregation after a transient mitotic arrest and the consequent formation of tetraploid G ₁ cells. These tetraploid cells are usually blocked to enter the subsequent S phase by the activation of p53/pRb pathway, which is referred to as the G₁ tetraploidy checkpoint. A human homologue of the Drosophila warts tumor suppressor, WARTS, is an evolutionarily conserved serine-threonine kinase and implicated in development of human tumors. We previously showed that WARTS plays a crucial role in controlling mitotic progression by forming a regulatory complex with zyxin, a regulator of actin filament assembly, on mitotic apparatus. However, when WARTS is activated during cell cycle and how the loss of WARTS function leads to tumorigenesis have not been elucidated. Here we show that WARTS is activated during mitosis in mammalian cells, and that overexpression of a kinase-inactive WARTS in Mat1 fibroblasts significantly induced mitotic delay. This delay resulted from prolonged activation of the spindle assembly checkpoint and was frequently followed by mitotic slippage and the development of tetraploidy. The resulting tetraploid cells then abrogated the G₁ tetraploidy checkpoint and entered S phase to achieve a DNA content of 8N. This impairment of G₁ tetraploidy checkpoint was caused as a consequence of failure to induce p53 expression by expressing a kinase-inactive WARTS. WARTS thus plays a critical role in maintenance of ploidy through its actions in both mitotic progression and the G₁ tetraploidy checkpoint.

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Cell-matrix interaction via CD44 is independently regulated by different metalloproteinases activated in response to extracellular Ca²⁺ influx and PKC activation

Nagano Osamu, Murakami Daizo, Hartmann Dieter, De Strooper Bart, Saftig Paul, Iwatsubo Takeshi, Nakajima Motowo, Shinohara Masanori, Saya Hideyuki

Journal of Cell Biology 165 ( 6 ) 893 - 902 2004.06

ISSN 0021-9525

CD44 is an adhesion molecule that interacts with hyaluronic acid (HA) and undergoes sequential proteolytic cleavages in its ectodomain and intramembranous domain. The ectodomain cleavage is triggered by extracellular Ca²⁺ influx or the activation of protein kinase C. Here we show that CD44-mediated cell-matrix adhesion is terminated by two independent ADAM family metalloproteinases, ADAM10 and ADAM17, differentially regulated in response to those stimuli. Ca²⁺ influx activates ADAM10 by regulating the association between calmodulin and ADAM10, leading to CD44 ectodomain cleavage. Depletion of ADAM10 strongly inhibits the Ca²⁺ influx-induced cell detachment from matrix. On the other hand, phorbol ester stimulation activates ADAM17 through the activation of PKC and small GTPase Rac, inducing proteolysis of CD44. Furthermore, depletion of ADAM10 or ADAM17 markedly suppressed CD44-dependent cancer cell migration on HA, but not on fibronectin. The spatio-temporal regulation of two independent signaling pathways for CD44 cleavage plays a crucial role in cell-matrix interaction and cell migration.

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Dependence of Paclitaxel Sensitivity on a Functional Spindle Assembly Checkpoint

Sudo Tamotsu, Nitta Masayuki, Saya Hideyuki, Ueno Naoto T.

Cancer Research 64 ( 7 ) 2502 - 2508 2004.04

ISSN 0008-5472

Paclitaxel stabilizes microtubules, causing mitotic arrest and activating the spindle assembly checkpoint. We determined whether suppression of the checkpoint genes Mad2 and BubR1 affects paclitaxel resistance and whether overexpression of Mad2 protein in checkpoint-defective cells enhances paclitaxel sensitivity. Suppression of Mad2 and BubR1 in paclitaxel-treated cancer cells abolished checkpoint function, resulting in paclitaxel resistance that correlated with suppression of cyclin-dependent kinase-1 activity. In contrast, overexpression of Mad2 in cells with a checkpoint defect attributable to low Mad2 expression restored checkpoint function, resulting in enhanced paclitaxel sensitivity that correlated with enhanced cyclin-dependent kinase-1 activity. However, overexpression of Mad2 failed to enhance paclitaxel sensitivity via checkpoint activation in Mad2-independent checkpoint-defective and -intact cells. Thus, checkpoint function is required for paclitaxel sensitivity. These findings show that any molecules that could interfere with the spindle assembly checkpoint could generate paclitaxel resistance in any patient.

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Autophosphorylation of a Newly Identified Site of Aurora-B Is Indispensable for Cytokinesis

Yasui Yoshihiro, Urano Takeshi, Kawajiri Aie, Nagata Koh Ichi, Tatsuka Masaaki, Saya Hideyuki, Furukawa Koichi, Takahashi Toshitada, Izawa Ichiro, Inagaki Masaki

Journal of Biological Chemistry 279 ( 13 ) 12997 - 13003 2004.03

ISSN 0021-9258

Mitotic kinases regulate cell division and its checkpoints, errors of which can lead to aneuploidy or genetic instability. One of these is Aurora-B, a key kinase that is required for chromosome alignment at the metaphase plate and for cytokinesis in mammalian cells. We report here that human Aurora-B is phosphorylated at Thr-232 through interaction with the inner centromere protein (INCENP) in vivo. The phosphorylation of Thr-232 occurs by means of an autophosphorylation mechanism, which is indispensable for the Aurora-B kinase activity. The activation of Aurora-B spatio-temporally correlated with the site-specific phosphorylation of its physiological substrates, histone H3 and vimentin. Overexpression of the TA mutant of Aurora-B, in which Thr-232 was changed into alanine, frequently induced multinuclearity in cells. These results indicate that the phosphorylation of Thr-232 is an essential regulatory mechanism for Aurora-B activation.

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Activation of m-Calpain Is Required for Chromosome Alignment on the Metaphase Plate during Mitosis

Honda Shinobu, Marumoto Tomotoshi, Hirota Toru, Nitta Masayuki, Arima Yoshimi, Ogawa Michio, Saya Hideyuki

Journal of Biological Chemistry 279 ( 11 ) 10615 - 10623 2004.03

ISSN 0021-9258

Calpains form a superfamily of Ca²⁺-dependent intracellular cysteine proteases with various isoforms. Two isoforms, μ- and m-calpains, are ubiquitously expressed and known as conventional calpains. It has been previously shown that the mammalian calpains are activated during mitosis by transient increases in cytosolic Ca²⁺ concentration. However, it is still unknown whether the activation of calpains contributes to particular events in mitosis. With the use of RNA interference (RNAi), we investigated the roles of calpains in mitosis. Cells reduced the levels of m-calpain, but not μ-calpain, arrested at prometaphase and failed to align their chromosomes at the spindle equator. Specific peptidyl calpain inhibitors also induced aberrant mitosis with chromosome misalignment. Although both m-calpain RNAi and calpain inhibitors affected neither the separation of centrosomes nor the assembly of bipolar spindles, Mad2 was detected on the kinetochores of the misaligned chromosomes, indicating that the prometaphase arrest induced by calpain inhibition is due to activation of the spindle assembly checkpoint. Furthermore, when calpain activity was inhibited in cells having monopolar spindles, chromosomes were clustered adjacent to the centrosome, suggesting that calpain activity is involved in a polar ejection force for metaphase alignment of chromosomes. Based on these findings, we propose that activation of m-calpain during mitosis is required for cells to establish the chromosome alignment by regulating some molecules that generate polar ejection force.

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Down-regulation of nuclear protein ICBP90 by p53/p21^Cip1/WAF1-dependent DNA-damage checkpoint signals contributes to cell cycle arrest at G1/S transition

Arima Yoshimi, Hirota Toru, Bronner Christian, Mousli Marc, Fujiwara Toshiyoshi, Niwa Shin Ichiro, Ishikawa Hiroyuki, Saya Hideyuki

Genes to Cells 9 ( 2 ) 131 - 142 2004.02

ISSN 1356-9597

Checkpoints, which monitor DNA damage and regulate cell cycle progression, ensure genomic integrity and prevent the propagation of transformed cells. DNA damage activates the p53-dependent checkpoint pathway that induces expression of p21^Cip1/WAF1, resulting in cell cycle arrest at G1/S transition by inhibition of cdk activity and DNA replication. ICBP90 was identified as a nuclear protein that binds to the TopoIIα gene promoter and is speculated to be involved in DNA replication. ICBP90 expression is cell cycle regulated in normal cells but stably high throughout cell cycle in various cancer cell lines. We here demonstrate that ICBP90 expression is down-regulated by the p53/p21^Cip1/WAF1-dependent DNA damage checkpoint signals. The reduction of ICBP90 appeared to be caused by both transcriptional suppression and protein degradation. Adenoviral expression of p21^Cip1/WAF1 directly led to ICBP90 reduction in p53^-/- HCT116 cells without DNA damage. Furthermore, ICPB90 depletion by RNA interference significantly blocked G1/S transition after DNA damage in HeLa cells. The down-regulation of ICBP90 is an important mechanism for cell cycle arrest at G1/S transition, which is induced by the activation of a p53/p21^Cip1/WAF1-dependent DNA-damage checkpoint. Deregulation of ICBP90 may impair the control of G1/S transition during checkpoint activation and lead to genomic instability.

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PKA phosphorylation and 14-3-3 interaction regulate the function of neurofibromatosis type I tumor suppressor, neurofibromin

Feng Liping, Yunoue Shunji, Tokuo Hiroshi, Ozawa Tatsuya, Zhang Dongwei, Patrakitkomjorn Siriporn, Ichimura Toru, Saya Hideyuki, Araki Norie

FEBS Letters 557 ( 1-3 ) 275 - 282 2004.01

ISSN 0014-5793

Neurofibromin, a neurofibromatosis type I (NF1) tumor suppressor gene product, has a domain acting as a GTPase activating protein and functions in part as a negative regulator of Ras. Loss of neurofibromin expression in NF1 patients is associated with elevated Ras activity and increased cell proliferation. Therefore, regulation of the function of neurofibromin is heavily involved in cell growth and differentiation. In the present study, we identified a novel cellular neurofibromin-associating protein, 14-3-3, which belongs to a highly conserved family of proteins that regulate intracellular signal transduction events in all eukaryotic cells. The interaction of 14-3-3 is mainly directed to the C-terminal domain (CTD) of neurofibromin, and the cAMP-dependent protein kinase (PKA)-dependent phosphorylation clustered on CTD-Ser (2576, 2578, 2580, 2813) and Thr (2556) is required for the interaction. Interestingly, the increased phosphorylation and association of 14-3-3 negatively regulate the function of neurofibromin. These findings indicate that PKA phosphorylation followed by 14-3-3 protein interaction may modulate the biochemical and biological functions of neurofibromin.

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Functional analysis of APC-Cdh1.

Sudo Tamotsu, Ueno Naoto T., Saya Hideyuki

Methods in molecular biology (Clifton, N.J.) 281 189 - 198 2004

ISSN 1064-3745

To maintain genomic integrity against various kinds of genotoxic stress, cells have multiple checkpoints in the cell cycle. When one of the cell cycle events, such as DNA synthesis, DNA repair, and chromosomal segregation, has not been successfully completed, checkpoints will delay progression until the step is correctly accomplished, and only then will they relieve the arrest to allow the cell to move to the next phase. Cells lacking functional checkpoints display genomic aberrations, resulting in the acquisition of phenotypic changes of cancer cells. Anaphase-promoting complex (APC) is activated by two regulatory proteins: Cdc20 and Cdh1. In yeast and Drosophila, Cdh1-dependent APC (APC-Cdh1) activity targets mitotic cyclins from the end of mitosis to the G1 phase. Loss of Cdh1 induces unscheduled accumulation of mitotic cyclins in G1, resulting in abrogation of G1 arrest caused by treatment with rapamycin, an inducer of p27Kip1. Furthermore, Cdh1-deficient DT40 cells fail to maintain DNA damage-induced G2 arrest, and Cdh1-APC is activated by X-irradiation-induced DNA damage. Thus, activation of Cdh1-APC plays a crucial role in both cdk inhibitor-dependent G1 arrest and DNA damage-induced G2 arrest. In light of the differences between normal and cancer cells, checkpoints can be ideal targets for developmental cancer therapeutics. In this chapter, we describe how to analyze three checkpoints (spindle assembly checkpoint, rapamycin-induced G1 checkpoint, and DNA damage-induced G2 checkpoint) in conjunction with cell synchronization.

Aurora-A Kinase Maintains the Fidelity of Early and Late Mitotic Events in HeLa Cells

Marumoto Tomotoshi, Honda Shinobu, Hara Toshihiro, Nitta Masayuki, Hirota Toru, Kohmura Eiji, Saya Hideyuki

Journal of Biological Chemistry 278 ( 51 ) 51786 - 51795 2003.12

ISSN 0021-9258

Aurora-A, a member of the Aurora/Ipl1-related kinase family, is overexpressed in various types of cancer and considered to play critical roles in tumorigenesis. To better understand the pathological effect of Aurora-A activation, it is first necessary to elucidate the physiological functions of Aurora-A. Here, we have investigated the roles of Aurora-A in mitotic progression with the small interfering RNA, antibody microinjection, and time lapse microscopy using human cells. We demonstrated that suppression of Aurora-A by small interfering RNA caused multiple events to fail in mitosis, such as incorrect separation of centriole pairs, misalignment of chromosomes on the metaphase plate, and incomplete cytokinesis. Antibody microinjection of Aurora-A into late G₂ cells induced dose-dependent failure in separation of centriole pairs at prophase, indicating that Aurora-A is essential for proper separation of centriole pairs. When we injected anti-Aurora-A antibodies into prometaphase cells that had separated their centriole pairs, chromosomes were severely misaligned on the metaphase plate, indicating that Aurora-A is required for proper movement of chromosomes on the metaphase plate. Furthermore, inhibition of Aurora-A at metaphase by microinjected antibodies prevented cells from completing cytokinesis, suggesting that Aurora-A also has important functions in late mitosis. These results strongly suggest that Aurora-A is essential for many crucial events during mitosis and that the phosphorylation of a series of substrates by Aurora-A at different stages of mitosis may promote diverse critical events in mitosis to maintain chromosome integrity in human cells.

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CENP-A phosphorylation by Aurora-A in prophase is required for enrichment of Aurora-B at inner centromeres and for kinetochore function

Kunitoku Naoko, Sasayama Takashi, Marumoto Tomotoshi, Zhang Dongwei, Honda Shinobu, Kobayashi Osamu, Hatakeyama Katsuyoshi, Ushio Yukitaka, Saya Hideyuki, Hirota Toru

Developmental Cell 5 ( 6 ) 853 - 864 2003.12

ISSN 1534-5807

The Aurora (Ipl1)-related kinases are universal regulators of mitosis. We now show that Aurora-A, in addition to Aurora-B, regulates kinetochore function in human cells. A two-hybrid screen identified the kinetochore component CENP-A as a protein that interacts with Aurora-A. Aurora-A phosphorylated CENP-A in vitro on Ser-7, a residue also known to be targeted by Aurora-B. Depletion of Aurora-A or Aurora-B by RNA interference revealed that CENP-A is initially phosphorylated in prophase in a manner dependent on Aurora-A, and that this reaction appears to be required for the subsequent Aurora-B-dependent phosphorylation of CENP-A as well as for the restriction of Aurora-B to the inner centromere in prometaphase. Prevention of CENP-A phosphorylation also led to chromosome misalignment during mitosis as a result of a defect in kinetochore attachment to microtubules. Our observations suggest that phosphorylation of CENP-A on Ser-7 by Aurora-A in prophase is essential for kinetochore function.

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Technetium-99m Sestamibi Single Photon Emission Computed Tomography Findings Correlated with P-glycoprotein Expression, Encoded by the Multidrug Resistance Gene-1 Messenger Ribonucleic Acid, in Intracranial Meningiomas

Saya Hideyuki, Yamada Kazuo

Neurologia Medico-Chirurgica 43 ( 12 ) 581 2003.12

ISSN 0470-8105

Prognostic Value of Epidermal Growth Factor Receptor in Patients with Glioblastoma Multiforme

Shinojima Naoki, Tada Kenji, Shiraishi Shoji, Kamiryo Takanori, Kochi Masato, Nakamura Hideo, Makino Keishi, Saya Hideyuki, Hirano Hirofumi, Kuratsu Jun Ichi, Oka Koji, Ishimaru Yasuji, Ushio Yukitaka

Cancer Research 63 ( 20 ) 6962 - 6970 2003.10

ISSN 0008-5472

Glioblastoma multiforme (GBM) frequently involves amplification and alteration of the epidermal growth factor receptor (EGFR) gene, resulting in overexpression of varied mutations, including the most common mutation, EGFRvIII, as well as wild-type EGFR (EGFRwt). To test the prognostic value of EGFR, we retrospectively analyzed the relationship between treatment outcomes and the EGFR gene in 87 newly diagnosed adult patients with supratentorial GBM enrolled in clinical trials. The EGFR gene status was assessed by Southern blots and EGFR expression by immunohistochemistry using three monoclonal antibodies (EGFR.25 for EGFR, EGFR.113 for EGFRwt, and DH8.3 for EGFRvIII). EGFR amplification was detected in 40 (46%) of the 87 GBM patients; in 39 (97.5%) of these, EGFR was overexpressed. On the other hand, in 46 of 47 patients without EGFR amplification (97.9%), no EGFR overexpression was present. There was a close correlation between EGFR amplification and EGFR overexpression (P < 0.0001). EGFRwt was overexpressed in 27 of the 40 (67.5%) patients with, and in none without, EGFR amplification (P < 0.0001). Similarly, EGFRvIII was overexpressed in 18 (45.0%) of 40 patients with and in 4 (8.5%) of 47 patients without EGFR amplification (P < 0.0001). The finding that 8 (20%) of the patients with EGFR amplification/EGFR overexpression manifested overexpression of neither EGFRwt nor EGFRvIII indicates that they overexpressed other types of EGFR. Multivariate analysis demonstrated that EGFR amplification was an independent, significant, unfavorable predictor for overall survival (OS) in all patients (P = 0.038, HR = 1.67). With respect to the relationship of age to EGFR prognostication, the EGFR gene status was a more significant prognosticator in younger patients, particularly in those <60 years (P = 0.0003, HR = 3.15), whereas not so in older patients. EGFRvIII overexpression, on the other hand, was not predictive for OS. However, in patients with EGFR amplification, multivariate analysis revealed that EGFRvIII overexpression was an independent, significant, poor prognostic factor for OS (P = 0.0044, HR = 2.71). This finding indicates that EGFRvIII overexpression in the presence of EGFR amplification is the strongest indicator of a poor survival prognosis. In GBM patients, EGFR is of significant prognostic value for predicting survival, and the overexpression of EGFRvIII with amplification plays an important role in enhanced tumorigenicity.

Preliminary observations on genetic alterations in pilocytic astrocytomas associated with neurofibromatosis 1

Tada Kenji, Kochi Masato, Saya Hideyuki, Kuratsu Jun Ichi, Shiraishi Shoji, Kamiryo Takanori, Shinojima Naoki, Ushio Yukitaka

Neuro-Oncology 5 ( 4 ) 228 - 234 2003.10

ISSN 1522-8517

Neurofibromatosis 1 (NF1) is an autosomal dominant disorder that predisposes sufferers to various forms of neoplasia. Among affected individuals, 15%-20% develop astrocytomas, especially pilocytic astrocytomas (PA), which are benign and classified as grade I by the World Health Organization. They are generally well circumscribed, and their progression is slow. NF1-associated PAs (NF1-PAs) occasionally behave as aggressive tumors. To elucidate underlying genetic events in clinically progressive NF1-PAs, we performed molecular genetic analysis on 12 PAs, including 3 NF1-PAs, for p53, p16, and epidermal growth factor receptor genes, as well as loss of heterozygosity (LOH) on chromosome 1p, 10, 17, and 19q. None of the obvious genetic alterations typically seen in higher grade astrocytomas were found in 9 sporadic PAs. However, in 2 of 3 NF1-PAs, microsatellite analysis showed LOH10, including the PTEN (phosphatase and tensin homolog deleted on chromosome 10) gene locus, despite the diagnosis of pilocytic astrocytoma; one of these also manifested homozygous deletion of the p16 gene. The other NF1-PA harbored only LOH of the NF1 gene locus (17q). Our preliminary results support the hypothesis that some NF1-PAs differ genetically from sporadic PAs.

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Aurora-A and an interacting activator, the LIM protein Ajuba, are required for mitotic commitment in human cells

Hirota Toru, Kunitoku Naoko, Sasayama Takashi, Marumoto Tomotoshi, Zhang Dongwei, Nitta Masayuki, Hatakeyama Katsuyoshi, Saya Hideyuki

Cell 114 ( 5 ) 585 - 598 2003.09

ISSN 0092-8674

Aurora family kinases contribute to regulation of mitosis. Using RNA interference in synchronized HeLa cells, we now show that Aurora-A is required for mitotic entry. We found that initial activation of Aurora-A in late G2 phase of the cell cycle is essential for recruitment of the cyclin B1-Cdk1 complex to centrosomes, where it becomes activated and commits cells to mitosis. A two-hybrid screen identified the LIM protein Ajuba as an Aurora-A binding protein. Ajuba and Aurora-A interact in mitotic cells and become phosphorylated as they do so. In vitro analyses revealed that Ajuba induces the autophosphorylation and consequent activation of Aurora-A. Depletion of Ajuba prevented activation of Aurora-A at centrosomes in late G2 phase and inhibited mitotic entry. Overall, our data suggest that Ajuba is an essential activator of Aurora-A in mitotic commitment.

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Hyaluronan oligosaccharides induce CD44 cleavage and promote cell migration in CD44-expressing tumor cells

Sugahara Kazuki N., Murai Toshiyuki, Nishinakamura Hitomi, Kawashima Hiroto, Saya Hideyuki, Miyasaka Masayuki

Journal of Biological Chemistry 278 ( 34 ) 32259 - 32265 2003.08

ISSN 0021-9258

CD44 is an adhesion molecule that serves as a cell surface receptor for several extracellular matrix components, including hyaluronan (HA). The proteolytic cleavage of CD44 from the cell surface plays a critical role in the migration of tumor cells. Although this cleavage can be induced by certain stimuli such as phorbol ester and anti-CD44 antibodies in vitro, the physiological inducer of CD44 cleavage in vivo is unknown. Here, we demonstrate that HA oligosaccharides of a specific size range induce CD44 cleavage from tumor cells. Fragmented HA containing 6-mers to 14-mers enhanced CD44 cleavage dose-dependently by interacting with CD44, whereas a large polymer HA failed to enhance CD44 cleavage, although it bound to CD44. Examination using uniformly sized HA oligosaccharides revealed that HAs smaller than 36 kDa significantly enhanced CD44 cleavage. In particular, the 6.9-kDa HA (36-mers) not only enhanced CD44 cleavage but also promoted tumor cell motility, which was completely inhibited by an anti-CD44 monoclonal antibody. These results raise the possibility that small HA oligosaccharides, which are known to occur in various tumor tissues, promote tumor invasion by enhancing the tumor cell motility that may be driven by CD44 cleavage.

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Neurofibromatosis type I tumor suppressor neurofibromin regulates neuronal differentiation via its GTPase-activating protein function toward Ras

Yunoue Shunji, Tokuo Hiroshi, Fukunaga Kohji, Feng Liping, Ozawa Tatsuya, Nishi Toru, Kikuchi Akira, Hattori Seisuke, Kuratsu Junichi, Saya Hideyuki, Araki Norie

Journal of Biological Chemistry 278 ( 29 ) 26958 - 26969 2003.07

ISSN 0021-9258

Neurofibromin, the neurofibromatosis type 1 (NF1) gene product, contains a central domain homologous to a family of proteins known as Ras-GTPase-activating proteins (Ras-GAPs), which function as negative regulators of Ras. The loss of neurofibromin function has been thought to be implicated in the abnormal regulation of Ras in NF1-related pathogenesis. In this study, we found a novel role of neurofibromin in neuronal differentiation in conjunction with the regulation of Ras activity via its GAP-related domain (GRD) in neuronal cells. In PC12 cells, time-dependent increases in the GAP activity of cellular neurofibromin (NF1-GAP) were detected after NGF stimulation, which were correlated with the down-regulation of Ras activity during neurite elongation. Interestingly, the NF1-GAP increase was due to the induction of alternative splicing of NF1-GRD type I triggered by the NGF-induced Ras activation. Dominant-negative (DN) forms of NF1-GRD type I significantly inhibited the neurite extension of PC12 cells via regulation of the Ras state. NF1-GRD-DN also reduced axonal and dendritic branching/extension of rat embryonic hippocampal neurons. These results demonstrate that the mutual regulation of Ras and NF1-GAP is essential for normal neuronal differentiation and that abnormal regulation in neuronal cells may be implicated in NF1-related learning and memory disturbance.

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Serum response factor is modulated by the SUMO-1 conjugation system

Matsuzaki Kazuhito, Minami Takeshi, Tojo Masahide, Honda Yoshiomi, Uchimura Yasuhiro, Saitoh Hisato, Yasuda Hideyo, Nagahiro Shinji, Saya Hideyuki, Nakao Mitsuyoshi

Biochemical and Biophysical Research Communications 306 ( 1 ) 32 - 38 2003.06

ISSN 0006-291X

Serum stimulation leads to activation of the serum response factor (SRF)-mediated transcription of immediate-early genes such as c-fos via various signal transduction pathways. We have previously reported that promyelocytic leukemia protein (PML) is involved in the transcriptional regulation by SRF. PML is one of the well-known substrates for modification by small ubiquitin-related modifier-1 (SUMO-1) and several SUMO-1-modified proteins associate with PML. Here, we report that SRF is modified by SUMO-1 chiefly at lysine¹⁴⁷ within the DNA-binding domain. Substitution of this target lysine for alanine did not affect the translocation of SRF to PML-nuclear bodies. The SRF mutant augmented the transcriptional activity under Rho A-stimulated condition but not under serum-starved condition, suggesting that activated SRF is suppressed by its sumoylation. These data support the transcriptional role of SUMO-1 conjugating system in cellular serum response.

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MCAF mediates MBD1-dependent transcriptional repression

Fujita Naoyuki, Watanabe Sugiko, Ichimura Takaya, Ohkuma Yoshiaki, Chiba Tsutomu, Saya Hideyuki, Nakao Mitsuyoshi

Molecular and Cellular Biology 23 ( 8 ) 2834 - 2843 2003.04

ISSN 0270-7306

DNA methylation is involved in a variety of genome functions, including gene control and chromatin dynamics. MBD1 is a transcriptional regulator through the cooperation of a methyl-CpG binding domain, cysteine-rich CXXC domains, and a transcriptional repression domain. A yeast two-hybrid screen was performed to investigate the role of MBD1 in methylation-based transcriptional repression. We report a mediator, MBD1-containing chromatin-associated factor (MCAF), that interacts with the transcriptional repression domain of MBD1. MCAF harbors two conserved domains that allow it to interact with MBD1 and enhancer-like transactivator Sp1. MCAF possesses a coactivator-like activity, and it seems to facilitate Sp1-mediated transcription. In contrast, the MBD1-MCAF complex blocks transcription through affecting Sp1 on methylated promoter regions. These data provide a mechanistic basis for direct inhibition of gene expression via methylation-dependent and histone deacetylation-resistant processes.

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Presenilin-dependent γ-secretase activity mediates the intramembranous cleavage of CD44

Murakami Daizo, Okamoto Isamu, Nagano Osamu, Kawano Yoshiaki, Tomita Taisuke, Iwatsubo Takeshi, De Strooper Bart, Yumoto Eiji, Saya Hideyuki

Oncogene 22 ( 10 ) 1511 - 1516 2003.03

ISSN 0950-9232

CD44 is the major adhesion molecule for the extracellular matrix components and is implicated in a wide variety of physiological and pathological processes including the regulation of tumor cell growth and metastasis. Our previous studies have shown that CD44 undergoes sequential proteolytic cleavages in the extracellular and transmembrane domains and the cleavage product derived from CD44 intramembranous cleavage acts as a signal transduction molecule. However, the underlying mechanism of the intramembranous cleavage of CD44 remains to be elucidated. In the present study, we report for the first time that CD44 is a substrate of the presenilin (PS)-dependent γ-secretase. We demonstrate that the intramembranous cleavage of CD44 induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment or mechanical scraping is blocked by γ-secretase inhibitors in U251MG cells and that this cleavage is also inhibited in PS-deficient mouse embryonic fibroblasts. Furthermore, we showed that PSI is redistributed to ruffling areas of the plasma membrane similarly to CD44 after TPA treatment, supporting our biochemical observation that PS1 is involved in the intramembranous cleavage of CD44. Our present findings suggest important implications for understanding CD44-dependent signal transduction and a potential role of PS/γ-secretase activity in the functional regulation of adhesion molecules.

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PML-nuclear bodies are involved in cellular serum response

Matsuzaki Kazuhito, Minami Takeshi, Tojo Masahide, Honda Yoshiomi, Saitoh Noriko, Nagahiro Shinji, Saya Hideyuki, Nakao Mitsuyoshi

Genes to Cells 8 ( 3 ) 275 - 286 2003.03

ISSN 1356-9597

Background: Serum stimulation leads to the activation of various signal transduction pathways in cells, and the resultant signals are integrated into the serum response factor (SRF)-dependent transcription of immediate-early genes such as c-fos. Results: To further characterize this response, we investigated the mechanism which controls serum response transcription in cultured human cells. Frequency of PML (promyelocytic leukaemia)-nuclear bodies (NBs) formation increases shortly after serum stimulation, probably facilitating the interaction of SRF and CBP acetyltransferase at the NBs. PML modulates SRF-mediated c-fos promoter activities upon addition of serum to cells or expression of constitutively active Rho family GTPases. We mapped the region in the SRF that interacts with PML to the C-terminal transactivation domain. An SRF mutant deleted of the transactivation domain neither co-localizes with CBP in NBs nor fulfills its transcriptional role. Under conditions of serum stimulation, the formation of NBs coincides with the immediate-early expression of the endogenous c-fos gene in fibroblasts and in all-trans retinoic acid-treated acute promyelocytic leukaemia NB4 cells. Conclusion: These data provide an insight into the involvement of NBs in modulating the transcription of serum-induced immediate-early genes.

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Tissue culture methods to study neurological disorders

Watabe Kazuhiko, Sakamoto Tsuyoshi, Kawazoe Yoko, Michikawa Makoto, Miyamoto Katsuichi, Yamamura Takashi, Saya Hideyuki, Araki Norie

Neuropathology 23 ( 1 ) 68 - 78 2003.03

ISSN 0919-6544

Previously, the authors have established spontaneously immortalized cell lines from long-term cultures of normal adult mouse Schwann cells. Establishment of such Schwann cell lines derived from murine disease models may greatly facilitate studies of the cellular mechanisms of their peripheral nervous system lesions in the relevant diseases. Recently, the authors have established immortalized Schwann cell lines derived from Niemann-Pick disease type C mice (NPC; spm/spm) and globoid cell leukodystrophy mice (twitcher). In the present study, long-term cultures were maintained of Schwann cells derived from dorsal root ganglia and consecutive peripheral nerves of another NPC mouse (npc^nih/npc^nih, npc^nih/+), myelin P0 protein-deficient mice (P0-/-, P0+/-) with their wild-type littermates (P0+/+), and neurofibromatosis type 1 gene (NF1)-deficient mice (Nf1^Fcr/+) for 8-10 months, and immortalized cell lines from all these animals established spontaneously. These cell lines had spindle-shaped Schwann cell morphology and distinct Schwann cell phenotypes and retained genomic and biochemical abnormalities, sufficiently representing the in vivo pathological features of the mutant mice. These immortalized Schwann cell lines can be useful in studies of nervous system lesions in these mutant mice and relevant human disorders.

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Caspase activation in neuronal and glial apoptosis following spinal cord injury in mice

Kuratsu Junichi, Kurisu Kaoro, Laws Edward R., Saya Hideyuki

Neurologia Medico-Chirurgica 43 ( 1 ) 29 - 30 2003.01

ISSN 0470-8105

The aryl hydrocarbon receptor nuclear transporter is modulated by the SUMO-1 conjugation system

Tojo Masahide, Matsuzaki Kazuhito, Minami Takeshi, Honda Yoshiomi, Yasuda Hideyo, Chiba Tsutomu, Saya Hideyuki, Fujii-Kuriyama Yoshiaki, Nakao Mitsuyoshi

Journal of Biological Chemistry 277 ( 48 ) 46576 - 46585 2002.11

ISSN 0021-9258

The aryl hydrocarbon receptor nuclear transporter (ARNT) is a member of the basic helix-loop-helix/PAS (Per-ARNT-Sim) family of transcription factors, which are important for cell regulation in response to environmental conditions. ARNT is an indispensable partner of the aryl hydrocarbon receptor (AHR) or hypoxia-inducible factor-1α. This protein is also able to form homodimers such as ARNT/ARNT. However, the molecular mechanism that regulates the transcriptional activity of ARNT remains to be elucidated. Here, we report that ARNT is modified by SUMO-1 chiefly at Lys²⁴⁵ within the PAS domain of this protein, both in vivo and in vitro. Substitution of the target lysine with alanine enhanced the transcriptional potential of ARNT per se. Furthermore, green fluorescent protein-fused ARNT tended to form nuclear foci in ∼20% of the transfected cells, and the foci partly colocalized with PML nuclear bodies. PML, one of the well known substrates for sumoylation, was found to augment the transcriptional activities of ARNT. ARNT bound AHR or PML, whereas the sumoylated form of ARNT associated with AHR, but not with PML, resulting in a reduced effect of PML, on transactivation by ARNT. Our data suggest that the sumoylation of ARNT modulates its transcriptional role through affecting the ability of ARNT to interact with cooperative molecules such as PML. This exemplifies a crucial role of protein sumoylation in modulating protein-protein interactions.

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Roles of aurora-A kinase in mitotic entry and G2 checkpoint in mammalian cells

Marumoto Tomotoshi, Hirota Toru, Morisaki Tetsuro, Kunitoku Naoko, Zhang Dongwei, Ichikawa Yasuko, Sasayama Takashi, Kuninaka Shinji, Mimori Tatsuyuki, Tamaki Norihiko, Kimura Masashi, Okano Yukio, Saya Hideyuki

Genes to Cells 7 ( 11 ) 1173 - 1182 2002.11

ISSN 1356-9597

Background: Various mitotic events are controlled by Cdc2-cyclin B and other mitotic kinases. Aurora/ Ipl1-related mitotic kinases were proved to play key roles in mitotic progression in diverse lower organisms. Aurora-A is a mammalian counterpart of aurora/Ipl1-related kinases and is thought to be a potential oncogene. However, the regulation of aurora-A activation and the commitment of aurora-A in the progression of G2-M phase are largely unknown in mammalian cells. Results: We demonstrated that aurora-A is activated depending on the activation of Cdc2-cyclin B in mammalian cells. Since Cdc2-cyclin B does not directly phosphorylate aurora-A, indirect pathways such as the inhibition of PP1 by Cdc2-cyclin B may act for the activation of aurora-A kinase. Microinjection of anti-aurora-A antibodies into HeLa cells at late G2 phase caused a significant delay in mitotic entry. Furthermore, aurora-A activation at G2-M transition was inhibited by DNA damage, and the over-expression of aurora-A induced the abrogation of the DNA damage-induced G2 checkpoint. Conclusions: Aurora-A is activated downstream of Cdc2-cyclin B and plays crucial roles in proper mitotic entry and G2 checkpoint control. Dysregulation of aurora-A induces abnormal G2-M transition in mammalian cells and may lead to chromosome instability, which results in the development and progression of malignant tumours.

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WARTS tumor suppressor is phosphorylated by Cdc2/cyclin B at spindle poles during mitosis

Morisaki Tetsuro, Hirota Toru, Iida Shin ichi, Marumoto Tomotoshi, Hara Toshihiro, Nishiyama Yasuyuki, Kawasuzi Michio, Hiraoka Takehisa, Mimori Tatsuyuki, Araki Norie, Izawa Ichiro, Inagaki Masaki, Saya Hideyuki

FEBS Letters 529 ( 2-3 ) 319 - 324 2002.10

ISSN 0014-5793

Identification of physiological substrates for Cdc2/cyclin B is crucial for understanding the functional link between mitotic events and Cdc2/cyclin B activation. A human homologue of the Drosophila warts tumor suppressor, termed WARTS, is a serine/threonine kinase and a dynamic component of the mitotic apparatus. We have found that Cdc2/cyclin B forms a complex with a fraction of WARTS in the centrosome and phosphorylates the Ser613 site of WARTS during mitosis. Immunocytochemical analysis has shown that the S613-phosphorylated WARTS appears in the spindle poles at prometaphase and disappears at telophase. Our findings suggest that Cdc/cyclin B regulates functions of WARTS on the mitotic apparatus.

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Proteomic analysis of human brain identifies α-enolase as a novel autoantigen in Hashimoto's encephalopathy

Ochi Hirofumi, Horiuchi Izumi, Araki Norie, Toda Tosifusa, Araki Tomohiro, Sato Kaori, Murai Hiroyuki, Osoegawa Manabu, Yamada Takeshi, Okamura Ken, Ogino Tomoaki, Mizumoto Kiyohisa, Yamashita Hirohumi, Saya Hideyuki, Kira Jun ichi

FEBS Letters 528 ( 1-3 ) 197 - 202 2002.09

ISSN 0014-5793

Hashimoto's encephalopathy (HE) is a rare autoimmune disease associated with Hashimoto's thyroiditis (HT). To identify the HE-related autoantigens, we developed a human brain proteome map using two-dimensional electrophoresis and applied it to the immuno-screening of brain proteins that react with autoantibodies in HE patients. After sequential MALDI-TOF-MASS analysis, immuno-positive spots of 48 kDa (pI 7.3-7.8) detected from HE patient sera were identified as a novel autoimmuno-antigen, α-enolase, harboring several modifications. Specific high reactivities against human α-enolase were significant in HE patients with excellent corticosteroid sensitivity, whereas the patients with fair or poor sensitivity to the corticosteroid treatment showed less reactivities than cut-off level. Although a few HT patients showed faint reactions to α-enolase, 95% of HT patients, patients with other neurological disorders, and healthy subjects tested were all negative. These results suggest that the detection of anti-α-enolase antibody is useful for defining HE-related pathology, and this proteomic strategy is a powerful method for identifying autoantigens of various central nervous system diseases with unknown autoimmune etiologies.

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Influence of p53 mutations on prognosis of patients with glioblastoma

Shiraishi Shoji, Tada Kenji, Nakamura Hideo, Makino Keishi, Kochi Masato, Saya Hideyuki, Kuratsu Jun Ichi, Ushio Yukitaka

Cancer 95 ( 2 ) 249 - 257 2002.07

ISSN 0008-543X

BACKGROUND. The influence of p53 mutations on the biology of astrocytic tumors is controversial. p53 is thought to be inactivated in the early stage of gliomagenesis; however, what role its inactivation plays in the malignancy of gliomas remains unknown. To understand the significance of p53 inactivation, the authors identified the locus of p53 gene mutation in glioma samples at different stages of progression and studied the correlation between the mutation and clinical behavior. METHODS. Samples from newly diagnosed gliomas, including pure and mixed astrocytomas, were analyzed for p53 mutations using a yeast functional assay. To determine the locus of the gene mutations, DNA sequencing was performed. RESULTS. The incidence of p53 mutations was higher in anaplastic astrocytomas (AA, 48%) than glioblastomas (GBM, 31%). There was no significant difference in the average ages of GBM patients with and without p53 mutations (54.9 years ± 2.3 and 53.2 years ± 4.6, respectively). In GBM patients, the mutation did not affect progression free survival or overall survival. Astrocytomas and GBM differed in the distribution of p53 mutation loci. CONCLUSIONS. The p53 gene mutation does not markedly affect the survival of GBM patients. The difference in the location of p53 mutations between AA and GBM suggests that in gliomas, the p53 mutation may contribute not only to tumorigenesis (as an early event) but also to progression to malignancy (as a late event).

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Cooperation of HECT-domain ubiquitin ligase hHYD and DNA topoisomerase II-binding protein for DNA damage response

Honda Yoshiomi, Tojo Masahide, Matsuzaki Kazuhito, Anan Tadashi, Matsumoto Mitsuhiro, Ando Masayuki, Saya Hideyuki, Nakao Mitsuyoshi

Journal of Biological Chemistry 277 ( 5 ) 3599 - 3605 2002.02

ISSN 0021-9258

Ubiquitin ligases define the substrate specificity of protein ubiquitination and subsequent proteosomal degradation. The catalytic sequence was first characterized in the C terminus of E6-associated protein (E6AP) and referred to as the HECT (homologous to E6AP C terminus) domain. The human homologue of the regulator of cell proliferation hyperplastic discs in Drosophila, designated hHYD, is a HECT-domain ubiquitin ligase. Here we show that hHYD provides a ubiquitin system for a cellular response to DNA damage. A yeast two-hybrid screen showed that DNA topoisomerase IIβ-binding protein 1 (TopBP1) interacted with hHYD. Endogenous hHYD bound the BRCA1 C-terminus domains of TopBP1 that are highlighted in DNA damage checkpoint proteins and cell cycle regulators. Using an in vitro reconstitution, specific E2 (ubiquitin-conjugating) enzymes (human UbcH4, UbcH5B, and UbcH5C) transferred ubiquitin molecules to hHYD, leading to the ubiquitination of TopBP1. TopBP1 was usually ubiquitinated and degraded by the proteosome, whereas X-irradiation diminished the ubiquitination of TopBP1 probably via the phosphorylation, resulting in the stable colocalization of up-regulated TopBP1 with γ-H2AX nuclear foci in DNA breaks. These results demonstrated that hHYD coordinated TopBP1 in the DNA damage response.

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Analysis of homozygous deletion of the p16 gene and correlation with survival in patients with glioblastoma multiforme

Kamiryo Takanori, Tada Kenji, Shiraishi Shoji, Shinojima Naoki, Nakamura Hideo, Kochi Masato, Kuratsu Jun Ichi, Saya Hideyuki, Ushio Yukitaka

Journal of Neurosurgery 96 ( 5 ) 815 - 822 2002

ISSN 0022-3085

Object. One of the most frequent genetic abnormalities found in patients with glioblastoma multiforme (GBM) is homozygous deletion of the p16 tumor suppressor gene. The authors investigated whether this deletion is associated with prognosis in patients with GBM. Methods. In 46 adult patients with supratentorial GBM, homozygous deletion of the p16 gene in tumor DNA was examined using the multiplex polymerase chain reaction assay. The deletion was confirmed in 14 (30.4%) of 46 patients, eight (30.8%) of 26 men and six (30.0%) of 20 women. Cox proportional hazard regression analysis, adjusted for age at surgery, the Karnofsky Performance Scale score, extent of resection, and the MIB-1 labeling index, revealed that homozygous deletion of the p16 gene was significantly associated with overall survival and progression-free survival in men, but not in women. Conclusions. The results of this study suggest that p16 homozygous deletion is a significant unfavorable prognostic factor in male patients with GBM.

Molecular mechanism for development of malignant tumors

Saya Hideyuki

Nishinihon Journal of Urology 64 ( 3 ) 127 - 131 2002

ISSN 0029-0726

Maintenace of genomic integrity following DNA damage depends upon the coordinated action of DNA repair and checkpoint systems. Checkpoints control a signaling system that changes the activity of cyclin-dependent kinases (cdks), resulting in delayed cell cycle progression. Arrest in G1 is thought to prevent aberrant replication of damaged DNA, while arrest in G2 allows cells to avoid segregation of defective chromosomes. The failure of the checkpoint system leads to genomic instability, which causes multiple DNA sequence alterations and chromosomal aberrations, resulting in a disposition to develop cancer. On the other hand, it has been elucidated that checkpoint abnormality is the major reason why cancer cells are sensitive to genotoxic agents, such as anti-cancer drugs and X-radiation. In normal cells, DNA damage activates checkpoints, resulting in cell cycle arrest. However, cancer cells which have checkpoint abnormalities cannot be arrested and the abrogation of the checkpoint system induces cell death via an unknown mechanism called "biochemical crush". Tumor cells which have more malignant phenotypes are considered to have defects in cell death signals which are induced by biochemical crush, and such cells are able to survive even with severe DNA damage. Accumulation of basic knowledge regarding checkpoint regulation and the cell death system is imortant for developing new therapeutic approaches for dealing with malignant tumors.

Hyperploidy induced by drugs that inhibit formation of microtubule promotes chromosome instability

Nitta Masayuki, Tsuiki Hiromasa, Arima Yoshimi, Harada Kei, Nishizaki Takahumi, Sasaki Kousuke, Mimori Tatsuyuki, Ushio Yukitaka, Saya Hideyuki

Genes to Cells 7 ( 2 ) 151 - 162 2002

ISSN 1356-9597

Background: Antimicrotubule drugs (AMDs), such as taxol and vincristine, are the most important addition to the chemotherapeutic armamentarium against human cancers. It has been shown that prolonged AMD treatment induces hyperploidy in G1-checkpoint-defective cancer cells and that these hyperploid cells subsequently undergo apoptosis. However, a fraction of these hyperploid cells are able to survive the prolonged mitotic stress and resume cell-cycle progression. Results: We established hyperploid clones that escaped from cell death after AMD treatment from two glioma cell lines, U251MG and U87MG. Subtractive comparative genomic hybridization (CGH) analysis revealed that clones derived from U87MG mainly had chromosome number changes, but that those from U251MG showed both numerical and structural chromosomal changes. Furthermore, numerous aberrations identified in U251MG clones were remarkably chromosome-specific, which may have been due to clonal selection for cells that have an advantage in growth and/or survival. All clones derived from both cell lines had abnormalities in chromosome segregation, and karyotypes of clones were more heterogeneous than those of parental cells, suggesting that cells having a higher chromosome number are subject to asymmetric chromosome segregation, resulting in a heterogeneous karyotype. All clones derived from U87MG and U251MG increased both centric and acentromeric micronuclei, suggesting the presence of chromosome structural abnormality. Conclusions: AMD treatment induces hyperploid formation and chromosome instability in checkpoint-deficient cancer cells.

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Usefulness of sampling with cotton swab for PCR-diagnosis of cutaneous leishmaniasis in the New World

Mimori T., Matsumoto T., Calvopiña M. H., Gomez E. A., Saya H., Katakura K., Nonaka S., Shamsuzzaman S. M., Hashiguchi Y.

Acta Tropica 81 ( 3 ) 197 - 202 2002

ISSN 0001-706X

In this study, we tested the polymerase chain reaction (PCR)-method to diagnose cutaneous leishmaniasis (CL) by taking exudate materials from lesions with cotton swabs, using our previously tested (PCR) panel comprised of Leishmania (Viannia) panamensis, L. (V.) braziliensis, L. (V.) guyanensis, L. (Leishmania) mexicana and L. (L.) amazonensis. The objectives of the present study were to improve the sampling method convenient for the patients and to test the usefulness of samples taken with cotton swabs. Sixteen patients were clinically diagnosed to have CL including one case of diffuse cutaneous leishmaniasis (DCL) in Ecuador and the causative Leishmania parasites were identified by PCR. All the 12 samples from CL patients of La Mana, positive for Leishmania DNA, were identified as L. (V.) panamensis, while two from CL of Huigra and one from DCL of San Ignacio were L. (L.) mexicana. In the field condition, taking biopsy material is not only painful but sometimes causes iatrogenic bacterial infections. Considering the sensitivity of the test, and convenient sampling procedure, it may be suggested that collection of exudates using cotton swabs may be a better alternative to biopsy sample for PCR-diagnosis of CL.

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Proteolytic cleavage of the CD44 adhesion molecule in multiple human tumors

Okamoto Isamu, Tsuiki Hiromasa, Kenyon Lawrence C., Godwin Andrew K., Emlet David R., Holgado-Madruga Marina, Lanham Irene S., Joynes Christopher J., Vo Kim T., Guha Abhijit, Matsumoto Mitsuhiro, Ushio Yukitaka, Saya Hideyuki, Wong Albert J.

American Journal of Pathology 160 ( 2 ) 441 - 447 2002

ISSN 0002-9440

Cell surface adhesion molecules are crucial for the development and/or pathogenesis of various diseases including cancer. CD44 has received much interest as a major adhesion molecule that is involved in tumor progression. We have previously demonstrated that the ectodomain of CD44 undergoes proteolytic cleavage by membrane-associated metalloproteases in various tumor cell lines. The remaining membrane-bound CD44 cleavage product can be detected using antibodies against the cytoplasmic domain of CD44 (anti-CD44cyto antibody). However, the cleavage of CD44 in primary human tumors has not been investigated. Using Western blots with anti-CD44cyto antibody to assay human tumor tissues, we show that the CD44 cleavage product can be detected in 58% (42 of 72) of gliomas but not in normal brain. Enhanced CD44 cleavage was also found in 67% (28 of 42) of breast carcinomas, 45% (5 of 11) of non-small cell lung carcinomas, 90% (9 of 10) of colon carcinomas, and 25% (3 of 12) of ovarian carcinomas. Tumors expressing a CD44 splice variant showed a significantly higher incidence of enhanced CD44 cleavage. The wide prevalence of CD44 cleavage suggests that it plays an important role in the pathogenesis of human tumors.

Study of autoantibodies against advanced glycation endproducts of the Maillard reaction

Araki Norie, Shibayama Rie, Ejima Yumiko, Nagai Ryoji, Araki Tomohiro, Saya Hideyuki, Horiuchi Seikoh

International Congress Series 1223 ( C ) 49 - 58 2001.12

ISSN 0531-5131

Long-term incubation of proteins with reducing sugar proceeds to advanced glycation end products (AGEs). We previously demonstrated the presence of AGE structures in human and animal tissues, and suggested the implication of AGEs in aging and diabetic complications. In this study, we tested whether AGEs present in vivo, such as N^ε-(carboxymethyl) lysine (CML) adduct, could serve as immunogens for generating autoantibodies. Plasma samples from STZ-induced diabetic rats reacted positively with AGE-proteins as well as CML-BSA, suggesting the presence of autoantibodies against AGE-structures, particularly CML in diabetic rats; the activity of the autoantibody increased with the duration of diabetic states, reflecting the accumulation of AGE proteins in these diabetic rats. An autoantibody fraction purified from the plasma of diabetic patients showed a positive reaction to AGE-proteins as well as to CML proteins, and also to human lens proteins which are known to undergo CML modification in vivo. Patients with renal failure caused by diabetes or nondiabetic pathologies had a higher autoantibody activity against AGE structures than that in normal subjects or diabetic patients without renal failure. These results suggest that AGEs accumulated in vivo serve as immunological epitopes for developing autoantibodies against AGEs, particularly against a CML structure, which might be used as an indicator of diabetic nephropathy or chronic renal failure.

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Proteolytic release of CD44 intracellular domain and its role in the CD44 signaling pathway

Okamoto Isamu, Kawano Yoshiaki, Murakami Daizo, Sasayama Takashi, Araki Norie, Miki Toru, Wong Albert J., Saya Hideyuki

Journal of Cell Biology 155 ( 5 ) 755 - 762 2001.11

ISSN 0021-9525

CD44 is a widely distributed cell surface adhesion molecule and is implicated in diverse biological processes. However, the nature of intracellular signaling triggered by CD44 remains to be elucidated. Here, we show that CD44 undergoes sequential proteolytic cleavage in the ectodomain and intracellular domain, resulting in the release of a CD44 intracellular domain (ICD) fragment. Consequently, CD44ICD acts as a signal transduction molecule, where it translocates to the nucleus and activates transcription mediated through the 12-O-tetradecanoylphorbol 13-acetate-responsive element, which is found in numerous genes involved in diverse cellular processes. Expression of an uncleavable CD44 mutant as well as metalloprotease inhibitor treatment blocks CD44-mediated transcriptional activation. In search of the underlying mechanism, we have found that CD44ICD potentiates transactivation mediated by the transcriptional coactivator CBP/p300. Furthermore, we show that cells expressing CD44ICD produce high levels of CD44 messenger RNA, suggesting that the CD44 gene is one of the potential targets for transcriptional activation by CD44ICD. These observations establish a novel CD44 signaling pathway and shed new light on the functional link between proteolytic processing of an adhesion molecule at the cell surface and transcriptional activation in the nucleus.

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Activation of Cdh1-dependent APC is required for G₁ cell cycle arrest and DNA damage-induced G₂ checkpoint in vertebrate cells

Sudo Tamotsu, Ota Yosuke, Kotani Shuji, Nakao Mitsuyoshi, Takami Yasunari, Takeda Shunichi, Saya Hideyuki

EMBO Journal 20 ( 22 ) 6499 - 6508 2001.11

ISSN 0261-4189

Anaphase-promoting complex (APC) is activated by two regulatory proteins, Cdc20 and Cdh1. In yeast and Drosophila, Cdh1-dependent APC (Cdh1-APC) activity targets mitotic cyclins from the end of mitosis to the G₁ phase. To investigate the function of Cdh1 in vertebrate cells, we generated clones of chicken DT40 cells disrupted in their Cdh1 loci. Cdh1 was dispensable for viability and cell cycle progression. However, similarly to yeast and Drosophila, loss of Cdh1 induced unscheduled accumulation of mitotic cyclins in G₁, resulting in abrogation of G₁, arrest caused by treatment with rapamycin, an inducer of p27^Kipl. Furthermore, we found that Cdh1^-1- cells fail to maintain DNA damage-induced G₂ arrest and that Cdh1-APC is activated by X-irradiation-induced DNA damage. Thus, activation of Cdh1-APC plays a crucial role in both cdk inhibitor-dependent G₁ arrest and DNA damage-induced G₂ arrest.

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Calpain-dependent proteolysis of merlin occurs by oxidative stress in meningiomas

Kaneko Takuro, Yamashima Tetsumori, Tohma Yasuo, Nomura Motohiro, Imajoh-Ohmi Shinobu, Saido Takaomi C., Nakao Mitsuyoshi, Saya Hideyuki, Yamamoto Hiroshi, Yamashita Junkoh

Cancer 92 ( 10 ) 2662 - 2672 2001.11

ISSN 0008-543X

BACKGROUND. The purpose of this study is to indicate that oxidative stress may contribute to occurrence of meningiomas. Recently, it was reported that aside from the neurofibromatosis type 2 (NF2) gene mutations, the calpain-dependent proteolysis of the NF2 gene product, merlin might be closely related to the development of certain NF2-related tumors. Although meningiomas are well known to occur more frequently in aged persons, it still remains unknown why calpain activation occurs predominantly in them. Because the production of free radicals with aging might be one of the causes of calpain activation especially in leptomeningeal cells being devoid of blood supply, the authors examined the relations between μ-calpain activation and merlin proteolysis induced by the oxidative stress. METHODS. The authors examined 12 patient-derived sporadic meningiomas and their primary cultured cells. Malignant glioma cell line (U-251MG), which had no relation to NF2, was used as a control. They were exposed to hydrogen peroxide (H₂O₂) for 1 hour. After oxidative stress, they were examined by Western blot and immunofluorescence microscopic analyses. RESULTS. Despite the consistent expressions of activated μ-calpain in 11 of 12 meningioma tissues, this calpain activation completely disappeared after culture; instead the full-length merlin appeared again in 8 of 11 cases. The treatment of cultured cells with hydrogen peroxide induced both μ-calpain-dependent cleavage of merlin and reduction of an intrinsic calpain inhibitor calpastatin. Such proteolysis was significantly blocked by a specific calpain inhibitor, Z-LLal. The full-length merlin was immunocytochemically colocalized with activated μ-calpain at the plasma membrane, and, after μ-calpain activation, the fragment of merlin translocated to the perinuclear cytoplasm or into the nucleus. CONCLUSIONS. These findings suggest that oxidative stress-induced activation of μ-calpain causes proteolysis of merlin conceivably to impair cell adhesion and/or contact inhibition of meningioma cells.

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Restoration of endogenous wild-type p53 activity in a glioblastoma cell line with intrinsic temperature-sensitive p53 induces growth arrest but not apoptosis

Ikeda Jun, Tada Mitsuhiro, Ishii Nobuaki, Saya Hideyuki, Tsuchiya Kazuhiko, Okaichi Kumio, Mishima Kazuhiko, Sawamura Yutaka, Fulci Giulia, Liu Ta Jen, Van Meir Erwin G.

International Journal of Cancer 94 ( 1 ) 35 - 43 2001.10

ISSN 0020-7136

p53 protein is a transcription factor involved in multiple tumor-suppressor activities including cell cycle control and apoptosis. TP53 gene is frequently mutated in glioblastoma, suggesting the importance of inactivation of this gene product in gliomagenesis. Restoration of p53 function in glioblastoma cell lines deficient for p53 has shown that p53 induces growth arrest or apoptosis depending on the cell line and vector used to transduce wild-type TP53 alleles. Considering that astrocytes grow and express p53, it is not clear whether these results reflect physiologic responses or the result of p53 overexpression in combination with cellular responses to viral vector infection. Here, we reassessed this issue using a glioblastoma cell line (LN382) that expresses an endogenous temperature-sensitive mutant p53. This cell line expresses TP53 alleles (100% as determined by a p53 transcriptional assay in yeast) mutated at codon 197 GTG (Val) > CTG (Leu). We found that the p53 protein in these cells acted as an inactive mutant at 37°C and as a functional wild-type p53 below 34°C as demonstrated by several lines of evidence, including (i) restoration of transactivating ability in yeast, (ii) induction of p53-modulated genes such as CDKN1^p21 and transforming growth factor-α, (iii) disappearance of accumulated p53 protein in the nucleus and (iv) decrease in steady state p53 protein levels. This temperature switch allowed p53 levels, which were close to physiological levels to dramatically reduce LN382 cell proliferation by inducing a G₁/S cell cycle block, but not to induce apoptosis. The lack of apoptosis was considered to be a result of the low level p53 expression, because increasing wild-type p53 levels by adenoviral-mediated gene transfer caused apoptosis in these cells. The LN382 cell line will be extremely useful for investigations into the roles of p53 in cellular responses to a variety of stimuli or damages.

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Phosphorylation of neurofibromin by cAMP-dependent protein kinase is regulated via a cellular association of N^G,N^G-dimethylarginine dimethylaminohydrolase

Tokuo Hiroshi, Yunoue Shunji, Feng Liping, Kimoto Masumi, Tsuji Hideaki, Ono Tomomichi, Saya Hideyuki, Araki Norie

FEBS Letters 494 ( 1-2 ) 48 - 53 2001.04

ISSN 0014-5793

The neurofibromatosis type 1 (NF1) tumor suppressor (neurofibromin) is thought to play crucial roles in cellular Ras- and cAMP-dependent kinase (PKA)-associated signals. In this study, we identified a cellular neurofibromin-associating protein, N^G,N^G-dimethylarginine dimethylaminohydrolase (DDAH) that is known as a cellular NO/NOS regulator. The interaction of DDAH was mainly directed to the C-terminal domain (CTD) and to the cysteine/serine-rich domain (CSRD) of neurofibromin, coinciding with the regions containing specific PKA phosphorylation sites. DDAH increased PKA phosphorylation of native neurofibromin in a dose-dependent manner, especially affecting the phosphorylation of CSRD. These findings suggest that the PKA accessibility of neurofibromin was regulated via DDAH interaction, and this regulation may modulate the cellular function of neurofibromin that is implicated in NF1-related pathogenesis.

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A novel tetracycline-dependent transactivator with E2F4 transcriptional activation domain.

Akagi K., Kanai M., Saya H., Kozu T., Berns A.

Nucleic Acids Research 29 ( 4 ) 2001.02

ISSN 1362-4962

A tetracycline-controlled gene expression system provides a powerful tool to dissect the functions of gene products. However, it often appears difficult to establish cell lines or transgenic animals stably expressing tetracycline-dependent transactivators, possibly as a result of toxicity of the transactivator domains used. In order to overcome this problem, we developed a novel tetracycline-dependent transactivator that works efficiently in mammalian cells. This transactivator is a fusion of the tet reverse repressor mutant and the transcriptional activating domain of human E2F4, which is ubiquitously expressed in vivo. We demonstrate here that this tetracycline-regulated gene expression system provides a two log transcriptional activation in mammalian cells as assessed by northern blot and luciferase analyses. Combining this system with green fluorescent protein reporter systems or microarray gene expression profiling will facilitate the study of gene function.

Mechanism of hyperploid cell formation induced by microtubule inhibiting drug in glioma cell lines

Tsuiki Hiromasa, Nitta Masayuki, Tada Mitsuhiro, Inagaki Masaki, Ushio Yukitaka, Saya Hideyuki

Oncogene 20 ( 4 ) 420 - 429 2001.01

ISSN 0950-9232

Checkpoint mechanism plays a crucial role in ensuring genomic integrity during cell cycle. Loss of checkpoint function is known to induce genomic instability and to alter ploidy of dividing cells. In this study, we examined mechanisms of hyperploid formation in glioma cells by treatment with nocodazole, which activates spindle assembly checkpoint by inhibiting microtubule polymerization. By prolonged nocodazole treatment, U251MG human glioma cell, which has a p53 mutation, underwent transient arrest at mitosis, and subsequently exited from mitotic arrest (termed 'mitotic slippage') followed by DNA replication without cytokinesis, resulting in hyperploid formation. Additionally, the heterogeneity in the number of centrosomes per cell increased during the hyperploid formation, suggesting that these hyperploid cells have genomic instability. By employing LN382 glioma cell that has a temperature-sensitive p53 mutation, we found that the activation of p53 prevents hyperploid formation after the prolonged nocodazole treatment. Furthermore, staurosporine, an inhibitor for a broad range of serine/threonine kinases including cdc2, was found to enhance hyperploid formation in U251MG cells by accelerating the induction of mitotic slippage. Interestingly, inhibitors specific for cdc2 kinase prevented the G2 to M transition but did not accelerate mitotic slippage, suggesting that staurosporinesensitive kinases other than cdc2 are required for maintenance of spindle assembly checkpoint. Moreover, the enhancement of hyperploid formation by staurosporine was also blocked by p53-dependent G1 checkpoint. These results suggest that abrogation of G1 checkpoint is a critical factor for formation of hyperploid cells after the mitotic slippage.

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Commentary

Laws E.R. Jr, Saya H., Ushio Y.

Neurologia Medico-Chirurgica 41 ( 5 ) 258 - 259 2001

ISSN 0470-8105

Identification of the cis-acting region in the NF2 gene promoter as a potential target for mutation and methylation-dependent silencing in schwannoma

Kino Takeshi, Takeshima Hideo, Nakao Mitsuyoshi, Nishi Toru, Yamamoto Keizo, Kimura Takahiro, Saito Yoshiki, Kochi Masato, Kuratsu Jun Ichi, Saya Hideyuki, Ushio Yukitaka

Genes to Cells 6 ( 5 ) 441 - 454 2001

ISSN 1356-9597

Background: Although mutational inactivation and allelic loss in the NF2 gene appear to be causal events in the majority of vestibular schwannomas, involvement of another potentially important mechanism, transcriptional inactivation, has not been investigated. Results: We cloned and functionally characterized the 5′-flanking region of the human NF2 gene and identified the molecular mechanisms that regulate NF2 expression. Luciferase assay and site-directed mutagenesis demonstrated that a 70-base pair (bp) region (-591 to -522 bp from the translation start site) was essential for the basic expression of the NF2 gene. A gel mobility shift assay indicated recognition by nuclear protein of the unusually long (≈66 bp) sequences in this region. Recognition was inhibited by either mutation of the binding core sequence or by methylation of three CpG sites. Point mutations at these CpG sites significantly decreased promoter activity, suggesting the importance of these sites. In 14 of 23 vestibular schwannomas, these three CpG sites were methylated in a site-specific manner and the methylation status was consistent with the expression of NF2 mRNA. Conclusions: Suppressed expression by aberrant methylation or mutation of the promoter elements could be an alternative mechanism for inactivation of the NF2 gene.

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Analysis of loss of heterozygosity on chromosome 10 in patients with malignant astrocytic tumors

Tada K., Shiraishi S., Kamiryo T., Nakamura H., Hirano H., Kuratsu J. I., Kochi M., Saya H., Ushio Y.

Journal of Neurosurgery 95 ( 4 ) 651 - 659 2001

ISSN 0022-3085

Object. The most frequent genetic abnormality in human malignant gliomas is loss of heterozygosity (LOH) on chromosome 10. Candidate genes on chromosome 10 that are associated with the prognosis of patients with anaplastic astrocytoma (AA) and glioblastoma (GBM) were evaluated. Methods. The authors used 12 fluorescent microsatellite markers on both arms of chromosome 10 to study LOH in 108 primary astrocytic tumors. The LOH on chromosome 10 was observed in 11 (32%) of 34 AAs and 34 (56%) of 61 GBMS. No LOH was detected in 13 low-grade gliomas. Loss of heterozygosity was not detected in any AA in the seven patients younger than 35 years, but it was discovered in 41% of the patients older than 35 years. The prognostic significance of LOH at each locus was evaluated in 89 patients older than 15 years; 33 (37%) had supratentorial AAs and 56 (63%) had supratentorial GBMs. The Cox proportional hazards model, adjusted for patient age at surgery, the preoperative Karnofsky Performance Scale score, and the extent of surgical resection revealed that LOH on marker D10S209 near the FGFR2 and DMBT1 genes was significantly associated with shorter survival in patients with AA. The LOH on markers D10S215 and D10S541, which contain the PTEN/MMAC1 gene between them, was significantly associated with shorter survival in patients with GBM. Conclusions. In the present study it is found that LOH on chromosome 10 is an age-dependent event for patients with AAs and that LOH on marker D10S209 near the FGFR2 and DMBT1 loci is a significantly unfavorable prognostic factor. It is also reported that LOH on the PTEN/MMAC1 gene is a significantly unfavorable prognostic factor in patients with GBM.

Expression of random peptide fused to invasin on bacterial cell surface for selection of cell-targeting peptides

Nakajima Hiroto, Shimbara Naoki, Shimonishi Yoko, Mimori Tatsuyuki, Niwa Shin Ichiro, Saya Hideyuki

Gene 260 ( 1-2 ) 121 - 131 2000.12

ISSN 0378-1119

The protein invasin expressed on the cell surface of the pathogenic bacteria Yersinia pseudotuberculosis mediates the entry of this bacterium into cultured mammalian cells. We have developed a system for expression of random peptides on the cell surface of Escherichia coli (E. coli) by creation of a fusion hybrid between a peptide and the invasin protein. The fusion protein constructs consist of part of the outer membrane domain of the invasin protein, six proline spacers, and a decamer of random peptides flanked by cysteine residues (CX₁₀C). Peptides were constitutively expressed on the cell surface in the resulting random decamer peptide library, which we designated as ESPEL (E. coli Surface Peptide Expression Library). The ESPEL was systematically screened for its binding affinity toward human cultured cells. Several bacterial clones were identified whose binding to human cells was mediated by peptides expressed on the bacterial cell surface. Flow cytometric analysis showed that both the identified bacterial clones and these corresponding chemically synthesized peptides bound to human cells specifically. The techniques described provide a new method that uses E. coli random peptide library to select targeting peptides for mammalian cells without any knowledge of the human cellular receptors.

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Molecular analysis of the h-warts/LATS1 gene in human breast cancer.

Morinaga N., Shitara Y., Yanagita Y., Koida T., Kimura M., Asao T., Kimijima I., Takenoshita S., Hirota T., Saya H., Kuwano H.

International Journal of Oncology 17 ( 6 ) 1125 - 1129 2000.12

ISSN 1019-6439

Loss of heterozygosity (LOH) on chromosome 6q is often observed in breast cancer, suggesting the existence of a putative tumor suppressor. Recently, a human homolog of the Drosophila warts tumor suppressor gene, h-warts/LATS1, was identified and mapped at chromosome 6q24-25.1. Mutation analysis of the h-warts/LATS1 was performed using 25 breast cancer tissues by RT-PCR SSCP analysis. Although LOH of the h-warts/LATS1 was found in one patient, no mutations were found. Two polymorphisms were found, but neither of them caused amino acid substitutions. Further investigations are necessary to elucidate the role of the h-warts/LATS1 gene in the carcinogenesis of breast cancer.

Ras oncoprotein induces CD44 cleavage through phosphoinositide 3-OH kinase and the Rho family of small G proteins

Kawano Yoshiaki, Okamoto Isamu, Murakami Daizo, Itoh Hiroshi, Yoshida Masaki, Ueda Shoichi, Saya Hideyuki

Journal of Biological Chemistry 275 ( 38 ) 29628 - 29635 2000.09

ISSN 0021-9258

CD44 is a cell surface adhesion molecule for several extracellular matrix components. We previously showed that CD44 expressed in cancer cells is proteolytically cleaved at the ectodomain through membrane-anchored metalloproteases and that CD44 cleavage plays a critical role in cancer cell migration. Therefore, cellular signals that promote the migration and metastatic activity of cancer cells may regulate the CD44 ectodomain cleavage. Here, we demonstrate that the expression of the dominant active mutant of Ha-Ras (Ha-Ras^Val-12) induces redistribution of CD44 to the newly generated membrane ruffling area and CD44 ectodomain cleavage. The migration assay revealed that the CD44 cleavage contributes to the Ha-Ras^Val-12-induced migration of NIH3T3 cells on hyaluronate substrate. Treatment with LY294002, an inhibitor for phosphoinositide 3-OH kinase (PI3K), significantly inhibits Ha-Ras^Val-12-induced CD44 cleavage, whereas that with PD98059, an inhibitor for MEK, does not. The active mutant p110 subunit of PI3K has also been shown to enhance the CD44 cleavage, suggesting that PI3K mediates the Ras-induced CD44 cleavage. Moreover, the expression of dominant negative mutants of Cdc42 and Rac1 inhibits the Ha-Ras^Val-12-induced CD44 cleavage. These results suggest that Ras > PI3K > Cdc42/Rac1 pathway plays an important role in CD44 cleavage and may provide a novel molecular basis to explain how the activated Ras facilitates cancer cell migration.

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Mapping and regulation of the tumor-associated epitope recognized by monoclonal antibody RS-11

Eto Hiroshi, Yoon Sam S., Bode Barrie P., Kamidono Sadao, Makino Keishi, Saya Hideyuki, Nakamura Hideo, Tanabe Kenneth K.

Journal of Biological Chemistry 275 ( 35 ) 27075 - 27083 2000.09

ISSN 0021-9258

We have previously described a rat monoclonal antibody, RS-11, which recognizes a tumor-associated antigen common to several species. In the present study, we have cloned and characterized the antigen recognized by RS-11. We screened a phage expression library prepared from HeLa cDNA and identified a clone that reacts with RS-11. DNA sequence analysis revealed that this clone contains sequences of keratin 18 (nucleotides 568-1196). We constructed several glutathione S-transferase fusion proteins and synthetic peptides based on this DNA sequence analysis and examined their reactivity with RS-11 to accurately map the RS-11 epitope. We determined that the epitope resides within a region of seven amino acids on the α-helix 2B domain of keratin 18 in which two amino acids (Leu³⁶⁶ and Lys³⁷⁰) are completely conserved among intermediate filaments as well as other keratin members that are immunoreactive with RS-11. These two residues are sequentially discontinuous but spatially adjacent. The RS-11 epitope is constitutively present in human primary cultured hepatocytes; however, its immunoreactivity with RS-11 is up-regulated by malignant transformation or stimulation with either epidermal growth factor or transforming growth factor alpha.

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Mechanism of transcriptional regulation by methyl-CpG binding protein MBD1

Fujita Naoyuki, Shimotake Nobuya, Ohki Izuru, Chiba Tsutomu, Saya Hideyuki, Shirakawa Masahiro, Nakao Mitsuyoshi

Molecular and Cellular Biology 20 ( 14 ) 5107 - 5118 2000.07

ISSN 0270-7306

MBD1 is a mammalian protein that binds symmetrically-methylated CpG sequences and regulates gene expression in association with DNA methylation. This protein possesses a conserved sequence, named methyl-CpG binding domain (MBD), among a family of methyl-CpG binding proteins that mediate the biological consequences of the methylation. In addition, MBD1 has at least five isoforms due to alternative splicing events, resulting in the presence of CXXC1, CXXC2, and CXXC3 in MBD1 isoforms v1 (MBD1v1) and MBD1v2, and CXXC1 and CXXC2 in MBD1v3 and -v4. In the present study, we have investigated the significance of MBD, CXXC, and the C-terminal transcriptional repression domain (TRD) in MBD1. A bacterially expressed MBD binds efficiently to densely methylated rather than to sparsely methylated DNAs. In both methylation-deficient Drosophila melanogaster SL2 cells and mammalian CHO-K1 cells, MB-D1v1 represses transcription preferentially from both unmethylated and sparsely methylated promoters, while MBD1v3 inhibits densely methylated but not unmethylated promoter activities. The CXXC3 sequence in MBD1v1 is responsible for the ability to bind unmethylated promoter. Furthermore, we have constructed mutant-type MBD1s in which the functionally important residues Arg22, Arg30, Asp32, Tyr34, Arg44, Ser45, and Tyr52 are changed to alanine to investigate the correlation between the structure and function of the MBD in MBD1. Excepting those for Ser45 and Tyr52, none of the recombinant MBD mutants bound to the densely methylated or unmethylated DNAs, and green fluorescent protein-fused MBD1 mutants did not localize properly in the nucleus. All the MBD1v1 and -v3 mutants lost the activity of methylation- dependent gene repression. Based on these findings we have concluded that MBD1 acts as a transcriptional regulator depending on the density of methyl- CpG pairs through the cooperation of MBD, CXXC, and TRD sequences.

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Zyxin, a regulator of actin filament assembly, targets the mitotic apparatus by interacting with h-warts/LATS1 tumor suppressor

Hirota Toru, Morisaki Tetsuro, Nishiyama Yasuyuki, Marumoto Tomotoshi, Tada Kenji, Hara Toshihiro, Masuko Norio, Inagaki Masaki, Hatakeyama Katsuyoshi, Saya Hideyuki

Journal of Cell Biology 149 ( 5 ) 1073 - 1086 2000.05

ISSN 0021-9525

The mitotic apparatus plays a pivotal role in dividing cells to ensure each daughter cell receives a full set of chromosomes and complement of cytoplasm during mitosis. A human homologue of the Drosophila warts tumor suppressor, h-warts/LATS1, is an evolutionarily conserved serine/threonine kinase and a dynamic component of the mitotic apparatus. We have identified an interaction of h-warts/LATS1 with zyxin, a regulator of actin filament assembly. Zyxin is a component of focal adhesion, however, during mitosis a fraction of cytoplasmic-dispersed zyxin becomes associated with h-warts/LATS1 on the mitotic apparatus. We found that zyxin is phosphorylated specifically during mitosis, most likely by Cdc2 kinase, and that the phosphorylation regulates association with h-warts/LATS1. Furthermore, microinjection of truncated h-warts/LATS1 protein, including the zyxin-binding portion, interfered with localization of zyxin to mitotic apparatus, and the duration of mitosis of these injected cells was significantly longer than that of control cells. These findings suggest that h-warts/LATS1 and zyxin play a crucial role in controlling mitosis progression by forming a regulatory complex on mitotic apparatus.

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Calpain-dependent proteolysis of NF2 protein

Kimura Yoriyoshi, Saya Hideyuki, Nakao Mitsuyoshi

Neuropathology 20 ( 3 ) 153 - 160 2000

ISSN 0919-6544

The neurofibromatosis type 2 (NF2) protein, known as merlin or schwannomin, is a tumor suppressor, and the NF2 gene has been found to be mutated in the majority of schwannomas and meningiomas, including both sporadically occurring and familial NF2 cases. Although the development of these tumors depends on the loss of merlin, the presence of tumors lacking detectable NF2 mutations suggests different mechanisms for inactivating merlin. Recent studies have demonstrated cleavage of merlin by calpain, a calcium-dependent neutral cysteine protease, and marked activation of the calpain system resulting in the degradation of merlin in these tumors. Increased turnover of merlin by calpain in some schwannomas and meningiomas exemplifies tumorigenesis linked to the calpain-mediated proteolytic pathway.

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Clinical significance of p53 functional loss in squamous cell carcinoma of the oropharynx

Obata Atsushi, Eura Masao, Sasaki Jiichiro, Saya Hideyuki, Chikamatsu Kazuaki, Tada Mitsuhiro, Iggo Richard D., Yumoto Eiji

International Journal of Cancer 86 ( 3 ) 187 - 193 2000

ISSN 0020-7136

We examined the frequency of p53 mutations in 38 oropharyngeal squamous cell carcinomas (SCC), using both a yeast functional assay and a conventional immunohistochemical staining method (IHC) to detect p53 mutations. We also explored the clinical importance of p53 mutations in oropharyngeal SCC. An accumulation of p53 protein was detected in 17 of the 38 (45%) tumors by IHC, whereas the yeast-based assay detected 6 additional p53 mutations, for a total of 23 tumors (61%) with p53 mutations. The cDNA sequencing analysis revealed that the 6 mutations undetected by IHC consisted of 3 frameshift, 1 nonsense and 2 missense mutations. Thus, the yeast functional assay was more sensitive than conventional IHC for detecting p53 mutations. Subsequently, the relationship between p53 mutations and the clinico-pathological parameters in oropharyngeal SCC was evaluated using the results of the functional assay. Mutation of p53 was not associated with the patient age, sex, tumor stage or degree of tumor cell differentiation. Interestingly, heavy drinking had a significant positive correlation with the p53 mutation, but heavy smoking did not, suggesting that prolonged exposure to alcohol is more related to p53 mutation in oropharyngeal SCC than to tobacco consumption. Radiation sensitivity was examined by comparing tumor size on magnetic resonance images before and after completion of therapy with 45 Gy radiation, in the 18 cases of T2 oropharyngeal SCC that were initially treated by radiotherapy. The results showed that tumors with wild-type p53 decreased in size significantly compared to those with mutant p53. In 33 patients treated with curative intent, the overall survival after the completion of therapy was better in patients with a wild-type p53 tumor than in patients with a mutant p53 tumor. We conclude that p53 mutation is associated with radiation resistance and a decreased probability of survival in oropharyngeal SCC. (C) 2000 Wiley-Liss. Inc.

Comparative analysis of brain proteins from p53-deficient mice by two- dimensional electrophoresis

Araki Norie, Morimasa Tadaomi, Sakai Tomoko, Tokuoh Hiroshi, Yunoue Shunji, Kamo Masaharu, Miyazaki Kenji, Abe Koji, Saya Hideyuki, Tsugita Akira

Electrophoresis 21 ( 9 ) 1880 - 1889 2000

ISSN 0173-0835

p53 is a tumor suppressor protein that regulates many cellular processes including the cell cycle, DNA repair, and apoptosis. It also serves as a critical regulator of neuronal apoptosis in the central nervous system (CNS). To elucidate the role of p53 in the CNS, brain proteins of p53 knock-out mice (p53-/-) were analyzed by two-dimensional gel electrophoresis (2-DE) and compared with those from p53 wild type (p53+/+) mice. Six types of brain tissue (temporal cortex, cerebellum, hippocampus, striatum, olfactory bulb, and cervical spinal cord) and other control tissues (lung and blood) from 18- week-old non-stress-induced mice were analyzed. The morphology of brains from p53-/- mice appeared to be normal and identical to that of p53+/+ mice, although lungs showed diffuse tumors that may have been caused by p53 deficiency. Comparative 2-D gel analysis showed that, on average, 7 of 886 spots from brain tissue were p53-/- specific, whereas 12 of 1008 spots from lung tissue were p53-/- specific. N-terminal amino acid sequence was determined for p53-/- specific proteins. In all brain tissues from p53-/- mice, a newly identified mouse mitochondrial NADH-ubiquinone oxidoreductase 24 kDa subunit showed decreased expression, and apolipoprotein A1 acidic forms showed increased expression. In addition, brain-type creatine kinase B chain and tubulin β-5 N-terminal fragment were increased in the p53-/- cerebellum, and a new protein in mouse, hydroxyacylglutathione hydrolase (glyoxalase II) was decreased in the temporal cortex of p53-/- mice. The alterations in protein expression identified in this study may imply a p53- related brain function. This is the first proteomic analysis on the p53-/- mouse brain, and further information based on this study will provide new insights into the p53 function in the CNS.

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NE-dlg, a mammalian homolog of Drosophila dlg tumor suppressor, induces growth suppression and impairment of cell adhesion

Hanada Norihisa, Makino Keishi, Koga Hisashi, Morisaki Tetsuro, Kuwahara Hiroaki, Masuko Norio, Tabira Yoichi, Hiraoka Takehisa, Kitamura Nobuo, Kikuchi Akira, Saya Hideyuki

International Journal of Cancer 86 ( 4 ) 480 - 488 2000

ISSN 0020-7136

Membrane-associated guanylate kinases (MAGUK's) are known to function as scaffolds for forming multiprotein complexes at the synaptic junctions of neuronal cells and at sites of epithelial cell-cell contact. In Drosophila, mutations of the lethal (I)-discs large (dig) gene, which encodes a MAGUK protein, leads to post-synaptic structure defects in neuronal cells and neoplastic overgrowth of epithelial cells. We previously showed that NE-dig (neuronal and endocrine dig), a human homolog of the dig, plays a crucial role in formation of synaptic structure in human neuronal cells. Here we demonstrate that NE-dig, similar to Drosophila dig, is involved in regulation of cell cycle progression and adhesive ability of non-neuronal cells. Overexpression of NE-dlg in proliferating cells including various cancer cell lines induced growth suppression and impairment of cell adhesive ability. Furthermore, NE-dlg overexpression caused the down-regulation of β-catenin in cancer cells regardless of mutations in the APC (adenomatous polyposis coil) gene. The PDZ domains of NE-dlg were found to be essential for the growth suppression, loss of adhesive property and down-regulation of β- catenin. We propose that NE-dlg regulates the cell growth and adhesive ability by controlling the level of β-catenin through an APC-independent pathway. Inactivation of NE-dlg may therefore contribute to development and/or progression of human neoplasms. (C) 2000 Wiley-Liss, Inc.

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Calcium influx triggers the sequential proteolysis of extracellular and cytoplasmic domains of E-cadherin, leading to loss of β-catenin from cell-cell contacts

Ito Kiyoharu, Okamoto Isamu, Araki Norie, Kawano Yoshiaki, Nakao Mitsuyoshi, Fujiyama Shigetoshi, Tomita Kimio, Mimori Tatsuyuki, Saya Hideyuki

Oncogene 18 ( 50 ) 7080 - 7090 1999.11

ISSN 0950-9232

Cadherins are major cell-cell adhesion molecules in both tumor and normal tissues. Although serum levels of soluble E-cadherin have been shown to be higher in the cancer patients than in healthy volunteers, the detail mechanism regulating release of soluble E-cadherin remains to be elucidated. Here we show that the ectodomain of E-cadherin is proteolytically cleaved from some cancer cells by a membrane-bound metalloprotease to yield soluble form, and the residual membrane-tethered cleavage product is subsequently degraded by intracellular proteolytic pathway. Futhermore, we show that extracellular calcium influx, that is induced by mechanical scraping of cells or ionomycin treatment, enhances the metalloprotease-mediated E-cadherin cleavage and the subsequent degradation of the cytoplasmic domain. Immunocytochemical analysis demonstrates that the sequential proteolysis of E-cadherin triggered by the calcium influx results in translocation of β-catenin from the cell-cell contacts to cytoplasm. Our data suggest that calcium influx-induced proteolysis of E-cadherin not only disrupts the cell-cell adhesion but also activates β-catenin-mediated intracellular signaling pathway, potentially leading to alterations in motility and proliferation activity of cells.

A point mutation of human p53, which was not detected as a mutation by a yeast functional assay, led to apoptosis but not p21(Waf1/Cip1/Sdi1) expression in response to ionizing radiation in a human osteosarcoma cell line, Saos-2

Okaichi Kumio, Wang Li Hong, Sasaki Ji ichiro, Saya Hideyuki, Tada Mitsuhiro, Okumura Yutaka

International Journal of Radiation Oncology Biology Physics 45 ( 4 ) 975 - 980 1999.11

ISSN 0360-3016

Purpose: The 123A point mutation of p53 showed increased radiosensitivity, whereas other mutations (143A, 175H, and 273H) were not affected. To determine the reason for increased radiosensitivity of the 123A mutation, the response of the transformant of 123A mutation to ionizing radiation (IR) was examined and compared to those of transformants with the wild type p53 or other point mutations (143A, 175H, and 273H).Methods and Materials: Stable transformants with a mutant or wild type p53 made by introducing cDNA into the human osteosarcoma cell line, Saos-2, which lacks an endogenous p53 were used. The transcriptional activity of mutant p53 was examined using a yeast functional assay. The transformants were examined for the accumulation of p53, the induction of p21(Waf1/Cip1/Sdi1) (hereafter referred to as p21), and the other response of p53-responsive genes (MDM2, Bax, and Bcl-2) by Western blotting. Apoptosis was analyzed by detection of DNA fragmentation.Results: The 123A point mutation of p53 was detected as a wild type in the yeast functional assay. The 123A mutant accumulated p53 in response to IR. The 123A mutant did not induce p21, but normally responded to MDM2, Bax, and Bcl-2. The 123A mutant entered apoptosis earlier than the wild type p53 transformant, and induced Fas at earlier in response to IR.Conclusion: The 123A mutant led to apoptosis, but not p21 expression in response to IR. The occurrence of apoptosis, but not induction of p21, corresponded to the radiosensitivity in the transformant. The early occurrence of apoptosis in 123A transformants may depend on the early induction of Fas. Copyright (C) 1999 Elsevier Science Inc.

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The stabilization mechanism of mutant-type p53 by impaired ubiquitination

Nagata Yoichi, Anan Tadashi, Yoshida Tetsuo, Mizukami Tamio, Taya Yoichi, Fujiwara Toshiyoshi, Kato Hirohisa, Saya Hideyuki, Nakao Mitsuyoshi

Oncogene 18 ( 44 ) 6037 - 6049 1999.10

ISSN 0950-9232

Mutant-type p53 (mt p53) is largely accumulated in cancer cells due to its increased stability. To elucidate the mechanism of mt p53 stabilization, we analysed the turnover of p53 mutated at codon 248 whose alteration is most frequently found in human cancers. Proteasome inhibition induced the accumulation of ubiquitinated mt p53, indicating that the ubiquitinated forms were essentially unstable and degraded by the proteasome. The presence of a small amount of the ubiquitinated mt p53 relative to the abundant non-ubiquitinated form suggested that the mt p53 ubiquitination was a rate-limiting process in the slow turnover. Two phenomena destabilizing mt p53 via the ubiquitin-proteasome degradation were proved to be independent. First, the coexpression of wild-type p53 (wt p53) promoted mt p53 destabilization as feedback regulation. Second, geldanamycin also induced mt p53 destabilization through the dissociation of the protein from hsp90 but not through the restoration of wt p53 function. Neither the mutant-specific conformation nor the N-terminal phosphorylation seemed to contribute directly to the mt p53 stabilization. Further, a two-dimensional gel electrophoresis revealed that most of the post-translationally modified mt p53 was equally subjected to ubiquitination and subsequent proteasomal degradation. These findings are that mt p53 stabilization depends on the ubiquitination due to both the loss of wt p53 function and the hsp90 association.

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A human homolog of Drosophila warts tumor suppressor, h-warts, localized to mitotic apparatus and specifically phosphorylated during mitosis

Nishiyama Yasuyuki, Hirota Toru, Morisaki Tetsuro, Hara Toshihiro, Marumoto Tomotoshi, Iida Shin Ichi, Makino Keishi, Yamamoto Hideyuki, Hiraoka Takehisa, Kitamura Nobuo, Saya Hideyuki

FEBS Letters 459 ( 2 ) 159 - 165 1999.10

ISSN 0014-5793

We identified a human homolog of Drosophila warts tumor suppressor gene, termed h-warts, which was mapped at chromosome 6q24-25.1. The h-warts protein has a serine/threonine kinase domain and is localized to centrosomes in interphase cells. However, it becomes localized to the mitotic apparatus, including spindle pole bodies, mitotic spindle, and midbody, in a highly dynamic manner during mitosis. Furthermore, h-warts is specifically phosphorylated in cells at mitotic phase, most likely by Cdc2 kinase. These findings suggest that h-warts functions as a component of the mitotic apparatus and is involved in proper progression of mitosis.

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Regulated CD44 cleavage under the control of protein kinase C, calcium influx, and the rho family of small G proteins

Okamoto Isamu, Kawano Yoshiaki, Matsumoto Mitsuhiro, Suga Moritaka, Kaibuchi Kozo, Ando Masayuki, Saya Hideyuki

Journal of Biological Chemistry 274 ( 36 ) 25525 - 25534 1999.09

ISSN 0021-9258

CD44 is a cell surface receptor for several extracellular matrix components and is implicated in tumor cell invasion and metastasis. Our previous studies have shown that CD44 expressed in cancer cells is proteolytically cleaved at the extracellular domain through membrane- associated metalloproteases and that CD44 cleavage plays a critical role in CD44-mediated tumor cell migration (Okamoto, I., Kawano, Y., Tsuiki, H., Sasaki, J., Nakao, M., Matsumoto, M., Suga, M., Ando, M., Nakajima, M., and Saya, H. (1999) Oncogene 18, 1435-1446). In the present study, we first demonstrate rapid degradation of the membrane-tethered CD44 cleavage product through intracellular proteolytic pathways, and it occurs only after CD44 extracellular cleavage. To address the mechanisms regulating CD44 cleavage at the extracellular domain, we show that 12-O-tetradecanoylphorbol 13-acetate (TPA) and the calcium ionophore ionomycin rapidly enhance metalloprotease- mediated CD44 cleavage in U251MG cells via protein kinase C-dependent and - independent pathways, respectively, suggesting the existence of multiple distinct pathways for regulation of CD44 cleavage. Concomitant with TPA- induced CD44 cleavage, TPA treatment induces redistribution of CD44 and ERM proteins (ezrin, radixin, and moesin) to newly generated membrane ruffling areas. Treatment with lysophosphatidic acid, which is known to activate the Rho-dependent pathway, inhibits TPA-induced CD44 redistribution and CD44 cleavage. Furthermore, overexpression of Rac dominant active mutants results in the redistribution of CD44 to the Rac-induced ruffling areas and the enhancement of CD44 cleavage. These results suggest that the Rho family proteins play a role in regulation of CD44 distribution and cleavage.

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Methylation-mediated transcriptional silencing in euchromatin by methyl- CpG binding protein MBD1 isoforms

Fujita Naoyuki, Takebayashi Shin Ichiro, Okumura Katsuzumi, Kudo Shinichi, Chiba Tsutomu, Saya Hideyuki, Nakao Mitsuyoshi

Molecular and Cellular Biology 19 ( 9 ) 6415 - 6426 1999.09

ISSN 0270-7306

DNA methylation of promoter-associated CpG islands is involved in the transcriptional repression of vertebrate genes. To investigate the mechanisms underlying gene inactivation by DNA methylation, we characterized a human MBD1 protein, one of the components of MeCP1, which possesses a methyl-CpG binding domain (MBD) and cysteine-rich (CXXC) domains. Four novel MBD1 isoforms (MBD1v1, MBD1v2, MBD1v3, and MBD1v4) were identified by the reverse transcription-PCR method. We found that these transcripts were alternatively spliced in the region of CXXC domains and the C terminus. Green fluorescent protein-fused MBD1 was localized to multiple foci on the human genome, mostly in the euchromatin regions, and particularly concentrated in the pericentromeric region of chromosome 1. Both the MBD sequence and genome methylation were required for proper localization of the MBD1 protein. We further investigated whether MBD1 isoforms are responsible for transcriptional repression of human genes. A bacterially expressed MBD1 protein bound preferentially to methylated DNA fragments containing CpG islands from the tumor suppressor genes p16, VHL, and E-cadherin and from an imprinted SNRPN gene. All MBD1 isoforms inhibited promoter activities of these genes via methylation. Interestingly, MBD1 isoforms v1 and v2 containing three CXXC domains also suppressed unmethylated promoter activities in mammalian cells. These effects were further manifested in Drosophila melanogaster cells, which lack genome methylation. Sp1-activated transcription of methylated p16 and SNRPN promoters was inhibited by all of the MBD1 isoforms, whereas the isoforms v1 and v2 reduced Sp1-activated transcription from unmethylated promoters as well. These findings suggested that the MBD1 isoforms have different roles in methylation-mediated transcriptional silencing in euchromatin.

A human homolog of Drosophila lethal(3)malignant brain tumor (l(3)mbt) protein associates with condensed mitotic chromosomes

Koga Hisashi, Matsui Shin Ichi, Hirota Toru, Takebayashi Shin Ichiro, Okumura Katsuzumi, Saya Hideyuki

Oncogene 18 ( 26 ) 3799 - 3809 1999.07

ISSN 0950-9232

The lethal(3)malignant brain tumor (D-l(3)mbt) gene is considered to be one of the tumor suppressor genes of Drosophila, and its recessive mutations are associated with malignant transformation of the neuroblasts in the larval brain. The structure of D-l(3)mbt protein is similar to Drosophila sex comb on midleg (Scm) protein which is a member of Polycomb group (PcG) proteins. We have isolated here the first human homolog of the D-l(3)mbt gene, designated h-l(3)mbt. Radiation hybrid mapping and fluorescence in situ hybridization (FISH) analysis localized the h-l(3)mbt gene to chromosome 20q12. The h-l(3)mbt transcript is expressed in most of the human adult normal tissues and cultured cell lines. However, some cancer cells markedly reduce the h-l(3)mbt protein expression. Immunocytochemical study revealed that the h-l(3)mbt protein shows a speckled and scattered distribution in interphase nuclei and completely associates with condensed chromosomes in mitotic cells. This subcellular localization has been shown to be different from that of Bmi1 protein which is a component of PcG complex. Furthermore, overexpression of h-l(3)mbt protein by using a Cre-mediated gene activation system leads to failures of proper chromosome segregation and cytokinesis, which result in formation of multinuclei in U251MG cells. These observations suggest that h-l(3)mbt protein has functions distinct from those of PcG proteins and may play a role in proper progression of cell division.

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Emerging therapeutic targets in schwannomas and meningiomas

Nakao Mitsuyoshi, Kimura Yoriyoshi, Saya Hideyuki

Expert Opinion on Therapeutic Targets 3 ( 2 ) 335 - 364 1999.06

ISSN 1472-8222

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Interaction of NE-dlg/SAP102, a neuronal and endocrine tissue-specific membrane-associated guanylate kinase protein, with calmodulin and PSD- 95/SAP90

Masuko Norio, Makino Keishi, Kuwahara Hiroaki, Fukunaga Kohji, Sudo Tamotsu, Araki Norie, Yamamoto Hideyuki, Yamada Yuji, Miyamoto Eishichi, Saya Hideyuki

Journal of Biological Chemistry 274 ( 9 ) 5782 - 5790 1999.02

ISSN 0021-9258

NE-dlg/SAP102, a neuronal and endocrine tissue-specific membrane- associated guanylate kinase family protein, is known to bind to C-terminal ends of N-methyl-D-aspartate receptor 2B (NR2B) through its PDZ (PSD- 95/Dlg/ZO-1) domains. NE-dlg/SAP102 and NR2B colocalize at synaptic sites in cultured rat hippocampal neurons, and their expressions increase in parallel with the onset of synaptogenesis. We have identified that NE-dlg/SAP102 interacts with calmodulin in a Ca²⁺-dependent manner. The binding site for calmodulin has been determined to lie at the putative basic α-helix region located around the src homology 3 (SH3) domain of NE-dlg/SAP102. Using a surface plasmon resonance measurement system, we detected specific binding of recombinant NE-dlg/SAP102 to the immobilized calmodulin with a K(d) value of 44 nM. However, the binding of Ca²⁺/calmodulin to NE-dlg/SAP102 did not modulate the interaction between PDZ domains of NE-dlg/SAP102 and the C- terminal end of rat NR2B. We have also identified that the region near the calmodulin binding site of NE-dlg/SAP102 interact with the GUK-like domain of PSD-95/SAP90 by two-hybrid screening. Pull down assay revealed that NE- dlg/SAP102 can interact with PSD-95/SAP90 in the presence of both Ca²⁺ and calmodulin. These findings suggest that the Ca²⁺/calmodulin modulates interaction of neuronal membrane-associated guanylate kinase proteins and regulates clustering of neurotransmitter receptors at central synapses.

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CD44 cleavage induced by a membrane-associated metalloprotease plays a critical role in tumor cell migration

Okamoto Isamu, Kawano Yoshiaki, Tsuiki Hiromasa, Sasaki Ji Ichiro, Nakao Mitsuyoshi, Matsumoto Mitsuhiro, Suga Moritaka, Ando Masayuki, Nakajima Motowo, Saya Hideyuki

Oncogene 18 ( 7 ) 1435 - 1446 1999.02

ISSN 0950-9232

CD44 is a cell surface receptor for hyaluronate, a component of the extracellular matrix (ECM). Although CD44 has been implicated in tumor invasion and metastasis, the molecular mechanisms remain to be elucidated. Here we find that CD44 expressed in cancer cells is cleaved at the membrane-proximal region of the ectodomain and the membrane-bound cleavage product can be detected using an antibody against the cytoplasmic domain of CD44. Furthermore, we report that CD44 cleavage is mediated by a membrane-associated metalloprotease expressed in cancer cells. A tissue inhibitor of metalloproteases-1 (TIMP-1), as well as metalloprotease inhibitors, inhibit CD44 cleavage in the cell-free assay. Contrary, serine protease inhibitors enhance CD44 cleavage, and the enhancement can be prevented by pretreatment with a metalloprotease inhibitor. Thus, CD44 cleavage is regulated by an intricate balance between some proteases and their inhibitors. Interestingly, treatment with the metalloprotease blocker 1,10-phenanthroline, which strongly prevent the CD44 cleavage, suppressed RERF-LC-OK lung cancer cell migration on a hyaluronate substrate, but not on several other substrates. These results suggest that CD44 cleavage plays a critical role in an efficient cell-detachment from a hyaluronate substrate during the cell migration and consequently promotes CD44-mediated cancer cell migration. Our present data indicate that CD44, not only ECM per se, is one of the targets of pericellular proteolysis involved in tumor invasion and metastasis.

Localization of the gene coding for ROCK II/Rho kinase on human chromosome 2p24

Takahashi N., Tuiki H., Saya H., Kaibuchi K.

Genomics 55 ( 2 ) 235 - 237 1999.01

ISSN 0888-7543

Serine/threonine kinase ROCK II/Rho kinase, which is an isozyme of ROCK I, is one of the targets for the small GTPase Rho. ROCK II regulates the formation of actin stress fibers and focal adhesions, cytokinesis, smooth muscle contraction, and the activation of c-fos serum response element. To elucidate the role of ROCK II in human disease, we have cloned a 4363-bp cDNA encoding human ROCK II. Sequencing analysis has shown that human ROCK II contains 1388 amino acid residues with a calculated molecular mass of approximately 161 kDa. Fluorescence in situ hybridization analysis showed that the human ROCK II gene is located on chromosome 2p24. Radiation hybrid mapping has shown that the human ROCK II gene is located very close to the highly polymorphic marker D2S359.

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Comparison of PCR results using scrape/exudate, syringe-sucked fluid and biopsy samples for diagnosis of cutaneous leishmaniasis in Ecuador

Matsumoto T., Hashiguchi Y., Gomez E. A., Calvopiña M. H., Nonaka S., Saya H., Mimori T.

Transactions of the Royal Society of Tropical Medicine and Hygiene 93 ( 6 ) 606 - 607 1999

ISSN 0035-9203

A novel human nucleoside diphosphate (NDP) kinase, Nm23-H6, localizes in mitochondria and affects cytokinesis

Tsuiki Hiromasa, Nitta Masayuki, Furuya Akiko, Hanai Nobuo, Fujiwara Toshiyoshi, Inagaki Masaki, Kochi Masato, Ushio Yukitaka, Saya Hideyuki, Nakamura Hideo

Journal of Cellular Biochemistry 76 ( 2 ) 254 - 269 1999

ISSN 0730-2312

Nucleoside diphosphate kinases (NDP kinases) are enzymes known to be conserved throughout evolution and have been shown to be involved in various biological events, in addition to the 'housekeeping' phosphotransferase activity. We present the molecular cloning of a novel human NDP kinase gene, termed Nm23- H6. Nm23-H6 gene has been mapped at chromosome 3p21.3 and is highly expressed in heart, placenta, skeletal muscle, and some of the cancer cell lines. Recombinant Nm23-H6 protein has been identified to exhibit functional NDP kinase activity. Immunolocalization studies showed that both endogenous and inducibly expressed Nm23-H6 proteins were present as short, filament-like, perinuclear radical arrays and that they colocalized with mitochondria. Cell fractionation study also demonstrated the presence of Nm23-H6 protein in a mitochondria- rich fraction. Moreover, induction of overexpression of Nm23-H6 in SAOS2 cells, using the Cre-loxP gene activation system, resulted in growth suppression and generation of multinucleated cells. Flow cytometric analysis also demonstrated that the proportion of cells with more than 4N DNA content increased to 28.1% after induction of Nm23-H6, coinciding with the appearance of multinucleated cells. These observations suggest that Nm23-H6, a new member of the NDP kinase family, resides in mitochondria and plays a role in regulation of cell growth and cell cycle progression.

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Cellular signal transduction via the neurofibromatosis type 2 tumor suppressor gene product; Merlin

Araki Norie, Saya Hideyuki

Seikagaku 71 ( 2 ) 128 - 134 1999

ISSN 0037-1017

The involvement of proteasome in myogenic differentiation of murine myocytes and human rhabdomyosarcoma cells

Mugita Norifumi, Honda Yoshiomi, Nakamura Hideo, Fujiwara Toshiyoshi, Tanaka Keiji, Omura Satoshi, Shimbara Naoki, Ogawa Michio, Saya Hideyuki, Nakao Mitsuyoshi

International Journal of Molecular Medicine 3 ( 2 ) 127 - 137 1999

ISSN 1107-3756

The murine C2C12 myocytes terminally differentiate to myotubes in the mitogen-depletion, and a portion of the cells undergo apoptosis. In this study, a specific proteasome inhibitor lactacystin induced cell cycle withdrawal and precocious expression of myosin in C2C12 cells in mitogen-enriched medium, but these cells did not fuse to form myotubes. Mitogen-starved myocytes could not differentiate to myotubes under the proteasome inhibition. The genes for p21, MyoD, Myogenin and RB were activated, and p27 gene was repressed under the proteasome inhibition, suggesting the transcriptional regulation of these genes linked to the proteasome activity. The induction of p21 prior to MyoD may contribute to the incomplete myogenesis in the presence of lactacystin. In addition, lactacystin-treated C2C12 cells did not undergo apoptosis, while proteasome accumulated in the nuclei of apoptotic cells but not in those of myotubes during mitogen-depleted differentiation. Further, lactacystin induced similarly incomplete differentiation in human RD embryonal rhabdomyosarcoma cells. Our findings demonstrated that proteasome has an essential role in myogenesis, especially in transcriptional control of myogenic and cell cycle regulators, cell fusion forming myotubes, and apoptosis.

Biological aspects of the neurofibromatosis type 2 gene product

Koga Hisashi, Takeshima Hideo, Araki Norie, Saya Hideyuki

Gann Monographs on Cancer Research 46 153 - 163 1999

ISSN 0072-0151

Neurofibromatosis 2 (NF2) is an inherited disorder characterized by a predisposition to multiple intracranial tumors. The protein encoded by the NF2 gene has striking similarities to ezrin, radixin and, moesin (ERM) proteins which link membrane proteins to the cytoskeleton. To elucidate the function of NF2 protein (merlin), we attempted to identify the merlin- binding proteins. The protein-binding assay detected five merlin-binding cellular proteins (MBPs). The entire ERM-homology domain of merlin was found to be essential for binding to all five proteins. Since most reported NF2 mutations were nonsense or frameshift mutations, we next performed a protein truncation test and found that 11 of 14 NF2 patients had truncational mutations (79%). Seven of the 11 patients (64%) had a splicing abnormality which leads to absence of exons in the ERM-homology domain. To examine the biological significance of the exon-missing mutations in the ERM-homology domain, we expressed the wild-type (wt-) and the various mutant (mu-) NF2s in a fibroblast cell line. The wt-NF2 showed intense punctate staining in the perinuclear cytoplasm in addition to overall staining of the submembranous area, whereas the mu-NF2s lacking exons in the ERM-homology domain showed granular staining at the perinuclear region without any accumulation at the submembrane region. Microinjection of wt-NF2 cDNA into the nucleus of fibroblasts revealed that wt-NF2 protein induced a progressive elongation of cell processes. Furthermore, cells that expressed mu-NF2 had decreased adhesion, which resulted in their detachment from the substratum. These findings suggest that the exon-missing mutations in the ERM-homology domain, through the loss of binding to MBPs, affect cell membrane-cytoskeleton signaling and consequently disrupt cell-to-cell or cell-to-matrix interaction.

Roles of Rho-associated kinase in cytokinesis; mutations in Rho- associated kinase phosphorylation sites impair cytokinetic segregation of glial filaments

Yasui Yoshihiro, Amano Mutsuki, Nagata Koh Ichi, Inagaki Naoyuki, Nakamura Hideo, Saya Hideyuki, Kaibuchi Kozo, Inagaki Masaki

Journal of Cell Biology 143 ( 5 ) 1249 - 1258 1998.11

ISSN 0021-9525

Rho-associated kinase (Rho-kinase), which is activated by the small GTPase Rho, regulates formation of stress fibers and focal adhesions, myosin fiber organization, and neurite retraction through the phosphorylation of cytoskeletal proteins, including myosin light chain, the ERM family proteins (ezrin, radixin, and moesin) and adducin. Rho-kinase was found to phosphorylate a type III intermediate filament (IF) protein, glial fibrillary acidic protein (GFAP), exclusively at the cleavage furrow during cytokinesis. In the present study, we examined the roles of Rho-kinase in cytokinesis, in particular organization of glial filaments during cytokinesis. Expression of the dominant-negative form of Rho-kinase inhibited the cytokinesis of Xenopus embryo and mammalian cells, the result being production of multinuclei. We then constructed a series of mutant GFAPs, where Rho-kinase phosphorylation sites were variously mutated, and expressed them in type III IF-negative cells. The mutations induced impaired segregation of glial filament (GFAP filament) into postmitotic daughter cells. As a result, an unusually long bridge-like cytoplasmic structure formed between the unseparated daughter cells. Alteration of other sites, including the cdc2 kinase phosphorylation site, led to no remarkable defect in glial filament separation. These results suggest that Rho-kinase is essential not only for actomyosin regulation but also for segregation of glial filaments into daughter cells which in turn ensures correct cytokinetic processes.

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Multifocal renal cell carcinoma

Miyake Hideaki, Nakamura Hideo, Hara Isao, Gohji Kazuo, Arakawa Soichi, Kamidono Sadao, Saya Hideyuki

Clinical Cancer Research 4 ( 10 ) 2491 - 2494 1998.10

ISSN 1078-0432

The reported incidence of satellite tumor lesions in kidneys resected by radical nephrectomy for renal cell carcinoma (RCC) is 7-25%; however, genetic analyses of satellite tumors in comparison with those of main tumor lesions have not been performed well. In the present study, we investigated the incidence of loss of heterozygosity (LOH) at chromosome arms 3p, 6q, 8p, 9p, 9q, and 14q using 18 microsatellite markers in 10 nonpapillary RCCs of 50 mm or less in diameter and the accompanying satellite tumor lesions to evaluate the genetic alterations in main and satellite tumors. LOH was detected in 10, 3, 5, 3, 2, and 3 cases at chromosome arms 3p, 6q, 8p, 9p, 9q, and 14q, respectively. In addition, primary and satellite tumor lesions in 8 of 10 cases exhibited identical patterns of LOH on the 18 loci examined. In the remaining two cases, both main and satellite tumors demonstrated LOH on the common seven and three loci, respectively, whereas for another locus, LOH was observed only in the satellite tumor lesions. The similarity of LOH patterns detected in main and satellite tumor lesions indicates that the presence of satellite tumors might be the result of intrarenal metastasis from the main tumor lesion. These findings strongly suggest that even in case of small nonpapillary RCC, nephron-sparing surgery might carry the risk of failing to prevent postoperative local recurrence due to the incomplete resection of unrecognized satellite tumors with genetic alterations similar to those of the main tumor.

Impairment of cell adhesion by expression of the mutant neurofibromatosis type 2 (NF2) genes which lack exons in the ERM-homology domain

Koga Hisashi, Araki Norie, Takeshima Hideo, Nishi Toru, Hirota Toru, Kimura Yoriyoshi, Nakao Mitsuyoshi, Saya Hideyuki

Oncogene 17 ( 7 ) 801 - 810 1998.08

ISSN 0950-9232

Neurofibromatosis 2 (NF2) is an inherited disorder characterized by a predisposition to multiple intracranial tumors. The protein encoded by the NF2 gene has striking similarities to ezrin, radixin and moesin (ERM) proteins which link membrane proteins to the cytoskeleton. Therefore, it can be speculated that the disruption of cytoskeletal organization by alterations in the NF2 gene is involved in the development of tumors. It has been reported that the majority of NF2 mutations were nonsense or frameshift mutations that result in premature termination of translation. To facilitate the detection of these mutations, we performed protein truncation test and found that 11 of 14 NF2 patients had truncational mutations (79%). Seven of the 11 patients (64%) had a splicing abnormality which lead to absence of exons in the ERM homology domain. To examine the biological significance of the exon-missing mutations in the ERM homology domain, we expressed the wild-type (wt-NF2) and the various mutant NF2s (mu-NF2s) in a fibroblast cell line by using both liposome-mediated transfection and nuclear microinjection of the expression plasmids. The wt-NF2 showed intense punctate staining in the perinuclear cytoplasm in addition to overall staining of the submembranous area, whereas the mu-NF2s lacking exons in the ERM homology domain showed granular staining at the perinuclear region without any accumulation at the submembrane region. Microinjection of wt-NF2 cDNA into the nucleus of VA13 cells revealed that wt-NF2 protein induced a progressive elongation of cell processes. Furthermore, cells that expressed mu-NF2 had decreased adhesion, which resulted in detachment from the substratum. These findings suggested that the exon-missing mutations in the ERM-homology domain may affect cell membrane-cytoskeleton signaling and consequently disrupt cell-to-cell or cell-to-matrix interaction.

Identification of a novel human homolog of the Drosophila dlg, P-dlg, specifically expressed in the gland tissues and interacting with p55

Nakamura Hideo, Sudo Tamotsu, Tsuiki Hiromasa, Miyake Hideaki, Morisaki Tetsuro, Sasaki Ji Ichiro, Masuko Norio, Kochi Masato, Ushio Yukitaka, Saya Hideyuki

FEBS Letters 433 ( 1-2 ) 63 - 67 1998.08

ISSN 0014-5793

We have identified a novel human homolog of the Drosophila dlg tumor suppressor gene, termed P-dlg, which has been mapped at chromosome 10q23. Unlike other human dlg homologs, P-dlg is expressed in placenta and various gland tissues but not in brain. The P-dlg protein is localized at the plasma membrane and cytoplasm, and it is expressed in the gland epithelial cells in normal prostate tissue but not in prostate cancer cell lines. Furthermore, we identified interaction between P-dlg and p55 palmitoylated membrane protein by yeast two-hybrid screening. These findings suggest that P-dlg forms a complex with p55 at the plasma membrane and plays roles in maintaining the structure of epithelial cells and transmitting extracellular signals to the membrane and cytoskeleton, which may negatively regulate cell proliferation. Copyright (C) 1998 Federation of European Biochemical Societies.

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The involvement of calpain-dependent proteolysis of the tumor suppressor NF2 (merlin) in schwannomas and meningiomas

Kimurau Yoriyoshi, Koga Hisashi, Araki Norie, Mugita Norifumi, Fujita Naoyuki, Takeshima Hideo, Nishi Tohru, Yamashima Tetsumori, Saido Takaomi C., Yamasaki Toshiki, Moritake Kouzo, Saya Hideyuki, Nakao Mitsuyoshi

Nature Medicine 4 ( 8 ) 915 - 922 1998.08

ISSN 1078-8956

Neurofibromatosis type 2 (NF2) protein, also known as merlin or schwannomin, is a tumor suppressor, and NF2 is mutated in most schwannomas and meningiomas. Although these tumors are dependent on NF2, some lack detectable NF2 mutations, which indicates that alternative mechanisms exist for inactivating merlin. Here, we demonstrate cleavage of merlin by the ubiquitous protease calpain and considerable activation of the calpain system resulting in the loss of merlin expression in these tumors. Increased proteolysis of merlin by calpain in some schwannomas and meningiomas exemplifies tumorigenesis linked to the calpain-mediated proteolytic pathway.

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Significant correlation of nitric oxide synthase activity and p53 gene mutation in stage I lung adenocarcinoma

Fujimoto Hisao, Sasaki Ji Ichiro, Matsumoto Mitsuhiro, Suga Moritaka, Ando Yukio, Iggo Richard, Tada Mitsuhiro, Saya Hideyuki, Ando Masayuki

Japanese Journal of Cancer Research 89 ( 7 ) 696 - 702 1998.07

ISSN 0910-5050

Nitric oxide (NO) and its derivatives can directly cause DNA damage and mutation in vitro and may play a role in the multistage carcinogenic process. It has been reported that NO induces mutation in the p53 tumor suppressor gene; we therefore analyzed the relationship between NO synthase (NOS) activity and p53 gene status in early-stage lung adenocarcinoma. Surgical samples were classified into two categories: 14 lung adenocarcinomas with high NOS activity (> 25 pmol/min/g tissue, category A), and 16 with low NOS activity (< 25 pmol/min/g tissue, category B). A yeast functional assay for p53 mutations disclosed a red colony that corresponded to a mutation in the p53 gene in 8 cases (57.1%) in category A and 3 cases (18.8%) in category B, the frequency being significantly higher in the former (P < 0.05). A p53 DNA sequence analysis revealed that 5 of the 8 p53 mutation-positive samples in category A had a G:C-to-T:A transversion, which is reported to he a major target of NO. The mechanism of carcinogenesis of adenocarcinoma is not fully understood, but these results suggest that an excess of endogenously formed NO may induce a p53 gene mutation containing mainly G:C-to-T:A transversion in the early stage of lung adenocarcinoma. Our results suggest that NO has potential mutagenic and carcinogenic activity, and may play important roles in human lung adenocarcinoma.

Association of the myosin-binding subunit of myosin phosphatase and moesin

Fukata Yuko, Kimura Kazushi, Oshiro Noriko, Saya Hideyuki, Matsuura Yoshiharu, Kaibuchi Kozo

Journal of Cell Biology 141 ( 2 ) 409 - 418 1998.04

ISSN 0021-9525

The small GTPase Rho is believed to regulate the actin cytoskeleton and cell adhesion through its specific targets. We previously identified the Rho targets: protein kinase N, Rho-associated kinase (Rho-kinase), and the myosin-binding subunit (MBS) of myosin phosphatase. We found that in MDCK epithelial cells, MBS accumulated at the tetradecanoylphorbol-13-acetate (TPA)-induced membrane ruffling area, where moesin, a member of the ERM (ezrin, radixin, and moesin) family, was localized. Neither membrane ruffling nor an accumulation of moesin and MBS at the free-end plasma membrane was induced when MDCK cells were stimulated with TPA after the microinjection of C3, which ADP-ribosylates and inactivates Rho. MBS was colocalized with moesin at the cell-cell contact sites in MDCK cells. We also found that moesin was coimmunoprecipitated with MBS from MDCK cells. Recombinant MBS interacted with the amino-terminal domains of moesin and ezrin. Myosin phosphatase composed of the catalytic subunit and MBS showed phosphatase activity toward moesin, which was phosphorylated by Rho-kinase. The phosphatase activity was inhibited when MBS was phosphorylated by Rho- kinase. These results suggest that MBS is recruited with moesin to the plasma membrane and that myosin phosphatase and Rho-kinase regulate the phosphorylation state of moesin downstream of Rho.

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Rapid identification of Leishmania species from formalin-fixed biopsy samples by polymorphism-specific polymerase chain reaction

Mimori Tatsuyuki, Sasaki Ji ichiro, Nakata Motomi, Gomez Eduardo A., Uezato Hiroshi, Nonaka Shigeo, Hashiguchi Yoshihisa, Furuya M. Masato, Saya Hideyuki

Gene 210 ( 2 ) 179 - 186 1998.04

ISSN 0378-1119

The precise identification and classification of Leishmania species is important for public health surveillance since different species cause different clinical features of the disease. A highly specific polymerase chain reaction (PCR) panel was developed to enable the identification of the five major Leishmania species that cause New World cutaneous leishmaniases. The primers used for this panel were designed to distinguish the polymorphism in sequences of commonly amplified DNA bands of the parasites produced by arbitrarily primed PCR. These polymorphism-specific PCR diagnoses were performed with formalin-fixed biopsy specimens of the leishmanial lesions from four patients in Ecuador and one hamster skin lesion, and these lesions were determined to be caused by Leishmania (Viannia) panamensis, L. (Leishmania) mexicana, and L. (L.) amazonensis. The PCR panel may offer an important and practical approach to the standardized identification of Leishmania species in field examinations.

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Neurosurgery and molecular biology

Nakamura Hideo, Saya Hideyuki

Neurological Surgery 26 ( 3 ) 200 - 206 1998.03

ISSN 0301-2603

Expression of CD44 splicing isoforms in lung cancers

Sasaki J. I Ichiro, Tanabe Kenneth K., Takahashi Kazuhisa, Okamoto Isamu, Fujimoto Hisao, Matsumoto Mitsuhiro, Suga Moritaka, Ando Masayuki, Saya Hideyuki

International Journal of Oncology 12 ( 3 ) 525 - 533 1998.03

ISSN 1019-6439

We examined CD44 isoform expression in 138 frozen tissue samples, which included primary lung carcinomas, adjacent non-tumorous lung tissues and benign lung diseases, by both reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemical analyses. CD44v8-10 mRNA and protein were dominantly expressed in non-small cell lung carcinomas (NSCLC), while non-tumorous tissues principally expressed CD44s and small cell lung carcinomas (SCLC) expressed either CD44s or no detectable CD44. These results indicate that CD44v8-10 is the dominant splicing isoform in NSCLC and can be practically utilized as a diagnostic marker and therapeutical target in NSCLC.

Identification of a human homolog of the Drosophila neuralized gene within the 10q25.1 malignant astrocytoma deletion region

Nakamura Hideo, Yoshida Mitsuhiro, Tsuiki Hiromasa, Ito Kiyoharu, Ueno Mikako, Nakao Mitsuyoshi, Oka Koji, Tada Mitsuhiro, Kochi Masato, Kuratsu Jun Ichi, Ushio Yukitaka, Saya Hideyuki

Oncogene 16 ( 8 ) 1009 - 1019 1998.02

ISSN 0950-9232

The loss of chromosome 10 is the most frequent genetic alteration found in malignant astrocytomas. In particular, the long arm of chromosome 10 was previously reported to have two or more common deletion regions where tumor suppressor genes may be located. In this study, we performed deletion mapping of 44 malignant astrocytomas using 12 microsatellite markers on chromosome 10q and demonstrated that the minimal common region of loss of heterozygosity (LOH) was present between D10S192 and D10S566 localized at 10q25.1. Subsequently, we have identified a novel gene, termed h-neu, within the region frequently deleted and found that h-neu encodes a protein with strong homology to the Drosophila neuralized (D-neu) protein. Northern blot and RT-PCR analyses revealed that h-neu mRNA was expressed at very low levels in human malignant astrocytoma tissues and the majority of glioma cell lines examined, while normal brains expressed h-neu transcript. Furthermore, DNA sequencing analysis of the h-neu transcript revealed one of the glioma cell lines, U251MG, had a single nucleotide substitution which resulted in an amino acid change from glycine (GGC) to serine (AGC) at codon 253. The D-neu gene is known to serve a critical function in neurogenesis in Drosophila, and loss-of-function mutations produce hyperplasia of primitive neuronal cells. These observations led us to hypothesize that h-neu gene plays a role in determination of cell fate in the human central nervous system and may act as a tumor suppressor whose inactivation could be associated with malignant progression of astrocytic tumors.

Overexpression of Bcl-2 in bladder cancer cells inhibits apoptosis induced by cisplatin and adenoviral-mediated p53 gene transfer

Miyake Hideaki, Hanada Norihisa, Nakamura Hideo, Kagawa Shunsuke, Fujiwara Toshiyoshi, Hara Isao, Eto Hiroshi, Gohji Kazuo, Arakawa Soichi, Kamidono Sadao, Saya Hideyuki

Oncogene 16 ( 7 ) 933 - 943 1998.02

ISSN 0950-9232

To investigate the effects of the expression of Bcl-2 protein in bladder cancer on the apoptosis induced by cisplatin or adenoviral-mediated p53 gene (Ad5CMV-p53) transfer, we transfected the bcl-2 gene into KoTCC-1, a human bladder cancer cell line that does not express the Bcl-2 protein. The Bcl-2-transfected KoTCC-1 (KoTCC-1/B) exhibited significantly higher resistance to both cisplatin and Ad5CMV-p53 transfer than did either the parental KoTCC-1 (KoTCC-1/P) or the vector-only transfected cell line (KoTCC-1/C). The flow cytometric analysis of the propidium iodide-stained nuclei and DNA fragmentation analysis after cisplatin or Ad5CMV-p53 treatment revealed DNA degradation in both KoTCC-1/P and KoTCC-1/C, whereas KoTCC1/B showed a marked inhibition of DNA degradation. Following the treatment with cisplatin or Ad5CMV-p53, the accumulation of p53 protein was highly detectable for a long period in KoTCC-1/B compared to that in KoTTC-1/P and KoTCC-1/C. Furthermore, the cisplatin and Ad5CMV-p53 treatments each reduced the volume of the subcutaneous tumors established in nude mice formed by KoTCC-1/P or KoTCC-1/C; in contrast, their reductive effects on the tumors formed by KoTCC-1/B were significantly suppressed. The intraperitoneal tumor cell implantation model revealed that the prognoses of mice injected with KoTCC-1/B were significantly inferior to those of the mice injected with either KoTCC-1/P or KoTCC-1/C after treatment with cisplatin or Ad5CMV-p53. These findings suggest that the expression of Bcl-2 in bladder cancer cells interferes with the therapeutic effects of cisplatin and Ad5CMV-p53 through the inhibition of the apoptotic pathway.

Molecular detection of cancer cells by competitive reverse transcription-polymerase chain reaction analysis of specific CD44 variant RNAs

Okamoto Isamu, Morisaki Tetsuro, Sasaki Ji Ichiro, Miyake Hideaki, Matsumoto Mitsuhiro, Suga Moritaka, Ando Masayuki, Saya Hideyuki

Journal of the National Cancer Institute 90 ( 4 ) 307 - 315 1998.02

ISSN 0027-8874

Background: CD44 is a cell surface glycoprotein implicated in such diverse biologic processes as lymphocyte activation and homing, extracellular matrix adhesion, and cellular migration. Primary transcripts of the CD44 gene can be alternatively spliced to produce a variety of messenger RNA (mRNA) species. The standard form of CD44 mRNA contains sequences from at least 20 genomic exons; variant mRNAs contain sequences from one or more additional exons (v1-10). Predominant expression of a specific CD44 variant, i.e., CD44v8-10, in several human carcinomas has been described previously. In this study, we developed a novel molecular approach for detecting cancer cells that overexpress CD44v8-10 mRNA. Methods: After finding that CD44v8-10 was predominantly expressed in non-small-cell lung and bladder carcinomas and that CD44v10 was predominantly expressed in leukocytes, we developed a competitive reverse transcription-polymerase chain reaction assay (CC-RT- PCR) that allows quantification of the relative expression of these two mRNA species in clinical specimens (i.e., determination of a v8-10/v10 ratio). CC- RT-PCR analysis was applied to pleural effusion specimens from patients with benign or malignant lung diseases as well as to spontaneously voided urine samples from patients with benign or malignant urologic diseases. Results: Fifty two of 54 samples from patients with benign diseases expressed CD44v10 predominantly (v8-10/v10 ratio ≤0.65), whereas 46 of 61 samples from patients with malignant diseases expressed CD44v8-10 predominantly (v8- 10/v10 ratio >1.00) (two-sided P<.001). CC-RT-PCR detected predominant expression of CD44v8-10 in cytologically negative samples from 11 patients who were later diagnosed with malignant disease. Conclusions: CC-RT-PCR analysis of CD44v8-10 expression could be an important adjunct to cytologic examination in cancer diagnosis, especially in detecting exfoliated cancer cells in pleural effusions and urine.

Evidence that HLA class II-restricted human CD4⁺ T cells specific to p53 self peptides respond to p53 proteins of both wild and mutant forms

Fujita Hiroshi, Senju Satoru, Yokomizo Hiroshi, Saya Hideyuki, Ogawa Michio, Matsushita Sho, Nishimura Yasuharu

European Journal of Immunology 28 ( 1 ) 305 - 316 1998.01

ISSN 0014-2980

By stimulating peripheral blood mononuclear cells of four healthy donors with a mixture of overlapping peptides representing the core domain of p53, we established two CD4⁺ αβ T cell clones and four lines that recognized wild-type and mutant p53 proteins as well as p53 self peptides in an HLA class II-restricted fashion. Two T cell lines established from two unrelated donors reacted to the p53 peptide (p)153-166 and p108-122, respectively, in the context of DP5 molecules. Two T cell clones established from two other unrelated donors were specific for p193-204 in the context of DRB1*1401 and for p153-165 in the context of DP5, respectively. These two T cell clones responded almost equally to both wild-type and four mutant recombinant p53 proteins. The proliferative responses of these T cell clones to p53 recombinant proteins were augmented by heat denaturing, thereby suggesting that altered conformation of the protein facilitates proteolytic processing to produce antigenic peptides. The DRB1*1401-restricted T cell clone specific for p193-204 killed a B lymphoblastoid cell line homozygous for HLA-DRB1*1401 when the cell line was pre-pulsed with p53 protein as well as peptide. These results indicate that CD4⁺ T cells reactive to p53 do exist in healthy individuals and the epitopes are probably ignored by the immune system under physiological conditions. It is suggested that such epitopes stimulate T cells to induce anti-p53 antibody production in cancer patients as previously reported by others. The possible involvement of p53-reactive T cells in anti-tumor immunity is discussed.

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Monitoring adenoviral p53 transduction efficiency by yeast functional assay

Tada M., Sakuma S., Iggo R. D., Saya H., Sawamura Y., Fujiwara T., Roth J. A.

Gene Therapy 5 ( 3 ) 339 - 344 1998

ISSN 0969-7128

Monitoring the transduction efficiency is of paramount importance in gene therapy. To monitor adenovirus-mediated wild-type p53 gene transfer, we have used a quantitative assay which tests the ability of human p53 to activate transcription in yeast. Selective amplification of cellular and viral p53 transcripts followed by quantitative assessment of mutant p53 content with the assay permits measurement of the wild-type p53 transduction efficiency into SF-188, U251MG and HUG31 glioblastoma cells. One reverse transcription primer tracks the wild-type/mutant ratio of endogenous p53 mRNA (P2), and the other the wild-type/mutant ratio of both endogenous and exogenous p53 mRNA (P1). Following infection of cell lines homozygous for mutant p53, the apparent transduction efficiency calculated (τ(o) = [P1 - P2]/[1 + P2]) correlated with the level of p21 expression. Transduction efficiency in heterozygous wild-type/mutant HUG31 cells increased linearly with multiplicity of infection (MOI) for τ(o) value, in keeping with theoretical predictions. These results suggest that the yeast p53 functional assay may be a useful tool for monitoring p53 gene therapy.

Highly specific and sensitive detection of malignancy in urine samples from patients with urothelial cancer by CD44v8-10/CD44v10 competitive RT-PCR

Miyake Hideaki, Okamoto Isamu, Hiara Isao, Gohji Kazuo, Yamanaka Kazuki, Arakawa Soichi, Kamidono Sadao, Saya Hideyuki

International Journal of Cancer 79 ( 6 ) 560 - 564 1998

ISSN 0020-7136

CD44 is a widely expressed cell surface adhesion molecule in which various isoforms arise from alternative RNA splicing mechanism. Overexpression of specific CD44 splice variant, i.e., CD44v8-10, has been found in several human malignancies and is considered to be associated with tumor progression and metastasis. We have demonstrated a novel molecular approach, CD44v8-10/CD44v10 competitive reverse transcription-polymerase chain reaction (CC-RT-PCR) for the detection of cancer cells overexpressing CD44v8-10 by the measurement of the transcriptional level of CD44v8-10 relative to that of CD44v10 (v8-10/v10 ratio). In this study, we initially examined the expression of CD44 splice variants in human urothelial cancers and their adjacent normal urinary tissues by RT-PCR. Any CD44 variant isoforms were barely detectable in normal urinary tissues, whereas CD44v8-10 was predominantly expressed in 23 of the 30 (77%) urothelial cancer specimens. We then applied CC-RT-PCR to spontaneously voided urine samples from patients with 80 urothelial cancer and 50 various benign urologic diseases. The CC-RTPCR analysis revealed that all of the samples associated with benign diseases presented a predominant expression of the CD44v10 transcript (the v8-10/v10 ratios = 0.00-0.87), whereas 62 of the 80 samples associated with urothelial cancers mostly expressed the CD44v8-10 transcript (the v8-10/v10 ratios > 1.00). In addition, the positivity rate obtained by the CC-RT-PCR analysis was high regardless of the pathological grade of the urothelial cancers, although the sensitivity of the cytological examination declined with decreasing tumor grade. Our findings suggest strongly that CC- RT-PCR is a non-invasive useful tool for the diagnosis of urine samples from patients with urothetial cancer.

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Interaction between CD44 and hyaluronic acid regulates human prostate cancer development

Miyake Hideaki, Hara Isao, Okamoto Isamu, Gohji Kazuo, Yamanaka Kazuki, Arakawa Soichi, Saya Hideyuki, Kamidono Sadao

Journal of Urology 160 ( 4 ) 1562 - 1566 1998

ISSN 0022-5347

Purpose: This study was designed to investigate the significance of the interaction between CD44 and hyaluronic acid in the development of human prostate cancer. Materials and Methods: We transfected the standard CD44 isoform (CD44s) cDNA into PC3, a human prostate cancer cell line that barely expresses CD44s protein. The effects of the reintroduction of CD44s into PC3 cells on the ability to bind hyaluronic acid (HA) were analyzed by the cell adhesion assay and by the cell migration assay. The in vitro growth rate of CD44s transfected PC3 was measured by using the MTT assay. We then evaluated the in vivo tumor development of CD44s transfected PC3 cells by subcutaneous, intravenous, and intraperitoneal injection models in athymic nude mice. Results: The introduction of CD44s in PC3 cells markedly enhanced the binding and migration of these cells to HA, but not to other extracellular matrix molecules. In vitro growth of CD44s-transfected PC3 was found to be significantly decreased. In addition, the CD44s-transfected PC3 cells also demonstrated reduced tumorigenicity and metastatic potential in in vivo experimental models. Conclusions: These findings suggest that the CD44s downregulation plays an important role in the development of human prostate cancer, in part through reduction of the ability to bind HA.

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Growth inhibition of experimental glioma by human interferon-β superinduced by cationic liposomes entrapping polyinosilic

Kuratsu J. I., Saya H., Tabuchi K.

Neurologia Medico-Chirurgica 38 ( 8 ) 473 - 474 1998

ISSN 0387-2572

Loss of merlin-p85 protein complex in NF2-related tumors

Takeshima Hideo, Nishi Toru, Yamamoto Keizo, Kino Takeshi, Nakamura Hideo, Saya Hideyuki, Kochi Masato, Kuratsu Jun Ichi, Ushio Yukitaka

International Journal of Oncology 12 ( 5 ) 1073 - 1078 1998

ISSN 1019-6439

The NF2 tumor suppressor gene product, designated merlin, belongs to the family of molecules that links membranous protein with the cytoskeleton. We have previously shown that merlin was co-immunoprecipitated with a cellular protein, p85, in cultured cell. To analyze the alteration of merlin and associated proteins in surgical specimens, we developed a new method for biotin-labeling of whole cellular proteins. Screening of tumor tissues using our method showed that none of malignant gliomas and half of the NF2-related tumors had altered p85 and merlin. Our detection method seems useful for the screening of merlin alterations in NF2-related tumors.

Molecular biology of tumor suppressor genes related to cell adhesion signalings

Saya H., Makino K., Kuwahara H., Masuko N., Nishiyama Y., Morisaki T., Sasaki J., Nakao M., Ushio Y.

International Journal of Pediatric Hematology/Oncology 5 ( 1 ) 44 - 45 1998

ISSN 1070-2903

Human ubiquitin-protein ligase Nedd4

Anan Tadashi, Nagata Yoichi, Koga Hisashi, Honda Yoshiomi, Yabuki Nami, Miyamoto Chikara, Kuwano Akira, Matsuda Ichiro, Endo Fumio, Saya Hideyuki, Nakao Mitsuyoshi

Genes to Cells 3 ( 11 ) 751 - 763 1998

ISSN 1356-9597

Background: Nedd4 is a ubiquitin-protein ligase containing a calcium/lipid-binding domain, multiple WW domains and a C-terminal Hect domain, which is required for both the ubiquitin transfer and the association with E2 ubiquitin-conjugating enzymes. Nedd4 has been reported to be involved in the selective ubiquitination of some regulatory proteins in transcription and membrane transport. Results: Three mRNA species for human Nedd4 were found to be 6.4-, 7.8- and 9.5-kb in size, and their expression patterns varied among normal tissues and cancer cell lines, indicating the tissue- and cell-specificities of Nedd4 expression. The Nedd4 protein, ≃120 kDa in weight, was found in the cytoplasm, mainly in the perinuclear region and cytoplasmic periphery, of human cultured cells. Neural differentiation induced not only the down-regulation of Nedd4 but also the localization of the protein to both the cytoplasm and neurites. To identify the ubiquitination pathway that is linked to Nedd4, we demonstrated that specific E2 enzymes, including human Ubc4, UbcH5B, UbcH5C, UbcH6 and UbcH7, could transfer ubiquitin molecules to Nedd4 at the active cysteine residue, whereas E6AP accepted ubiquitins from Ubc4, UbcH5B, UbcH5C and UbcH7. Furthermore, nuclear localization of N-terminal deletion mutant Nedd4 enabled us to investigate the interaction between Nedd4 and E2 enzyme (Ubc4 or UbcH7) in the cell. The simultaneous expression of the full-length Nedd4 and E2 enzyme revealed that both proteins mostly colocalized in the cytoplasmic periphery, while the N-terminal deleted Nedd4 induced the nuclear and perinuclear colocalization with E2 enzyme. Conclusion: Our findings suggested that Nedd4 plays an important role in the cell regulation, including neural differentiation through cooperation with specific E2 ubiquitination pathways.

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Identification of a novel, tissue-specific calpain htra-3; a human homologue of the Caenorhabditis elegans sex determination gene

Mugita Norifumi, Kimura Yoriyoshi, Ogawa Michio, Saya Hideyuki, Nakao Mitsuyoshi

Biochemical and Biophysical Research Communications 239 ( 3 ) 845 - 850 1997.10

ISSN 0006-291X

We cloned a novel human gene encoding a tissue-specific calpain, termed htra-3, which is highly homologous to the tra-3 sex determination gene of Caenorhabditis elegans. The predicted htra-3 polypeptide had similarity to the calpain large subunits in domain organization throughout domains I to III, but the sequences of domain IV lacked calcium-binding motifs. Northern blot analysis revealed high expression in the colon, small intestine and testis. Radiation hybrid mapping localized the htra-3 gene to chromosome 11q14 (2.53cR apart from WI-3895). Western blot analysis demonstrated that the approximately 73-kDa htra-3 protein was transiently expressed in COS-7 cells. These observations, together with the genetic information in C. elegans, suggest a unique function for htra-3 protein.

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Alternative splicing of the erythropoietin receptor gene correlates with erythroid differentiation in rat hematopoietic and leukemic cells

Fujita Masayuki, Takahashi Rei, Kitada Kazuhiro, Watanabe Rei, Kitazawa Sohei, Ashoori Faramarz, Liang Ping, Saya Hideyuki, Serikawa Tadao, Maeda Sakan

Cancer Letters 112 ( 1 ) 47 - 55 1997.01

ISSN 0304-3835

An alternative splicing of the rat erythropoietin receptor (EpoR) gene; was identified in normal and erythroleukemia cells. A 105 bp insert was found at a region corresponding to the extracellular domain of EpoR. The alternative transcript was translated to a soluble EpoR (EpoR-S) expressed in spleen, bone marrow, and cultured erythroleukemia cells in addition to the full-length EpoR (EpoR-F). One of the rat erythroleukemia sublines, K4DT, which partially lost erythroid phenotypes and manifested monocyte/macrophage characteristics also lacked EpoR-S expression. Thus, expression of EpoR-S may play an important role in differentiation of rat erythroid cells.

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Role of alternative splicing of the rat erythropoietin receptor gene in normal and erythroleukemia cells

Fujita M., Takahashi R., Liang P., Saya H., Ashoori F., Tachi M., Kitazawa S., Maeda S.

Leukemia 11 ( SUPPL. 3 ) 444 - 445 1997

ISSN 0887-6924

An alternatively splicing of the rat erythropoietin receptor (EpoR) gene was identified in normal and erythroleukemia cells by the reverse transcription PCR method. Insertion of a 105 bp fragment at the region corresponding to the extracellular domain of rat EpoR was found. The insert sequence, which encodes additional 21 amino acids, is similar to that previously found in the mouse EpoR gene, however, has an additional 27 bp direct repeat. Due to the presence of a stop codon in the insert, the alternative transcript is translated to a truncated and soluble form of EpoR which is preferentially expressed in liver, spleen, kidney, heart, and bone marrow cells as well as cultured erythroleukemia cells. These findings suggest that alternative splicing of the EpoR gene may play an important role in proliferation and differentiation of rat erythroid cells.

Cloning and characterization of NE-dlg

Makino Keishi, Kuwahara Hiroaki, Masuko Norio, Nishiyama Yasuyuki, Morisaki Tetsurou, Sasaki Ji Ichiro, Nakao Mitsuyoshi, Kuwano Akira, Nakata Motomi, Ushio Yukitaka, Saya Hideyuki

Oncogene 14 ( 20 ) 2425 - 2433 1997

ISSN 0950-9232

We have cloned a cDNA for a novel human homolog of the Drosophila discs large (dlg) tumor suppressor protein, termed NE-dlg (neuronal and endocrine dlg). Northern blot analysis revealed that the gene is highly expressed in neuronal and endocrine tissues. Fluorescence in situ hybridization (FISH) and radiation hybrid mapping studies localized the NE-dlg gene to chromosome Xq13. We also found that the NE-dlg gene encoded a 100 kDa protein. Immunolocalization studies using an NE-dlg antibody showed that the protein tended to be expressed in non-proliferating cells, such as neurons, cells in Langerhans islets of the pancreas, myocytes of the heart muscles, and the prickle and functional layer cells of the esophageal epithelium. Proliferative cells, including various cultured cancer cell lines and basal cells in the esophageal epithelium, showed little expression of the NE-dlg protein. In addition, yeast two-hybrid screening and in vitro binding assays revealed that the NE-dlg interacted with the carboxyl-terminal region of the APC tumor suppressor protein. These data suggest that NE-dlg negatively regulates cell proliferation through its interaction with the APC protein.

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Human nedd-4 as a family member of ubiquitin ligases

Anan Tadashi, Nagata Yoichi, Honda Yoshiomi, Kimura Yoriyoshi, Kuwano Akira, Nomura Nobuo, Matsuda Ichiro, Saya Hideyuki, Nakao Mitsuyoshi

Japanese Journal of Human Genetics 42 ( 1 ) 38 1997

ISSN 0916-8478

Ubiquitin-proteasome proteolytic pathway is involved in a number of cellular physiology. In this system, a ubiquitin ligase, or E3 enzyme, is a major component which recognizes a specific substrate protein for targeted degradation. E6-associated protein (E6-AP) has been recently found to be a member of the E3 enzyme family that is characterized by a conserved carboxy-terminal sequence, termed Aectf-domain. Using human EST data bases, we identified five uncharacterized genes of this family. By FISH and radiation hybrid panel these genes were mapped to subchromosomal locations as follows; E6-AP (15ql2-13), NEDD-4 (15q22; D15S209), D28476 (2q35-36; D2S396), D13635 (7q36; FB16B4),and D25215 (4q21-22; WI-6336). NEDD-4 gene was mapped close to the translocation breakpoint of acute promyelocytic leukemia. Interestingly, NEDD-4 was highly conserved among species from yeast to human, suggesting its significant function on basic biomodulation. Northern blot analysis showed that NEDD-4 mRNA was much higher expressed in lung carcinoma A549 and chronic myelogenous leukemia K562 than in other cancer cell lines tested. The rest of the members, except D25215, were ubiquitously expressed in all cells. In immunoblot analysis, human NEDD-4 protein was down-regulated during neural differentiation in BDNFtreated neuroblastoma cells. Collectively, our data suggest that NEDD-4 protein may function as a ubiquitin ligase closely related to development, differentiation and tumorigenesis.

Neurofibromatosis type 2 (NF2)

Araki N., Takeshima H., Saya H.

Japanese Journal of Cancer and Chemotherapy 24 ( 11 ) 1427 - 1431 1997

ISSN 0385-0684

Neurofibromatosis type 2 (NF2) is an autosomal dominantly inherited disorder, strongly associated with development of benign intracranial tumors, including bilateral vestibular schwannomas and meningiomas. The NF2 gene located on the human chromosome 22q12 was recently cloned, and the protein it encodes termed merlin/schwannomin was found to be strikingly similar to the moesin-ezrin-radixin (MER) family of cytoskeleton-associated proteins. Mutations of NF2 gene have been found not only in the NF2 patient but also in NF2-related sporadic tumors such as acoustic schwannomas and meningiomas, suggesting that the NF2 gene functionally acts as a tumor suppressor gene. To elucidate the biological function of merlin, we have detected five merlin binding cellular proteins. The N-terminal region of merlin, the entire merlin-ezrin-radixin-moesin (MERM) homology domain, was found to be essential for to all five binding proteins. Since most reported NF2 mutations in the region were determined to be necessary for binding, the mutations probably impair binding. The majority of NF2 mutations are nonsense mutations or frameshifts that resulted in premature termination of merlin. The lack of these interactions caused by such mutations of NF2 gene may affect the cellular signals, resulting in benign intracranial tumors in NF2 patients.

Functional analysis of tumor suppressor p53 protein regulated by ubiquitin-proteasome system

Nagata Yoichi, Anan Tadashi, Honda Yoshiomi, Kimura Reiji, Saya Hidevuki, Nakao Mitsuvoshi, Nakata Motomi, Shinbara Naoki, Tanaka Keiii, Omura Satoshi

Japanese Journal of Human Genetics 42 ( 1 ) 38 1997

ISSN 0916-8478

The tumor suppressor p53 protein is most important for cell regulation such as cell cycle and apoptosis. In HPV-infected cells including HeLa cells, a viral oncpprptein E6 and a cellular E6-associated protein (E6AP) function together as a ubiquitin ligase targetting for p53. The complex plays a major role in the tumorigenesis by rapid turnover of p53. However, the mechanism of p53 degradation in normal cells remains unclear. 1) Using proteasome inhibitors, p53 protein was significantly accumulated in primary nbroblasts as well as HeLa cells in vivo. 2) Recombinant p53 proteins destined for wild- or mutant(273His)-type were analysed in an appropriate buffer and rabbit reticulpcyte lysates in vitro. Much slower degradation of mutant-p53 was found compared with the case of wild-p53. Interestingly, bpth-types of p53 were multiubiquitinated in a similar manner. 3) In fibroblasts infected with p53-expressing adenovirus, wild-p53 protein was co-immunoprecipitated with anti-E6AP antibody. In summary, E6AP may also work in the p53 ubiquitination in normal cells adding to E6-dependent case. Uoiguitinated mutant-p53 becomes resistant to degradation, suggesting that mutant-p53 is hardly recognized and/or degraded by proteasome due to unknown mechanism.

Neurofibromatosis 2 gene has novel alternative splicings which controls intracellular protein binding

Nishi Toru, Takeshima Hideo, Hamada Kazuya, Yoshizato Kimio, Koga Hisashi, Sato Kyoichi, Yamamoto Keizo, Kitamura Isao, Kochi Masato, Kuratsu Jun Ichi, Saya Hideyuki, Ushio Yukitaka

International Journal of Oncology 10 ( 5 ) 1025 - 1029 1997

ISSN 1019-6439

Three novel isoforms of the neurofibromatosis 2 (NF2) gene transcripts generated from alternative splicing were identified from normal human brain, schwannoma and glioma tissues. The 3 novel transcripts lack exon 2, exons 2 and 3, exons 2-4, respectively. Recombinant isoform proteins encoded by those new transcripts have lost the previously reported ability to bind ³⁵S-methionine labeled cellular proteins. Two of seven glioblastoma tissues expressed significantly high levels of the shorter transcripts whereas low grade astrocytomas expressed levels similar to those found in normal brain, suggesting that genomic mutation or aberrant alternative splicing of the NF2 gene may contribute to the progression of malignant gliomas.

Molecular biology of malignant brain tumors

Saya H.

Japanese Journal of Neurosurgery 6 ( 5 ) 306 - 310 1997

ISSN 0917-950X

Molecular genetic analyses have revealed that the genesis and progression of tumors are the result of accumulated changes in positive and negative growth regulatory genes. Several sequential genetic alterations are considered to be required to direct cells toward the malignant phenotype. Recent studies have demonstrated that glial oncogenesis is also involved in multiple, but relatively consistent genetic alterations. The most frequent chromosomal alterations observed in astrocytic tumors include amplification of chromosome 7 and losses on chromosomes 9p, 10, and 17p. The p53 gene and the CDKN2/p16 gene have been indicated as target genes for losses of chromosome 17p and 9p, respectively. The biochemical characters of these tumor suppressor proteins are discussed in this review.

Tight-binding inhibitory sequences against pp60(c-src) identified using a random 15-amino-acid peptide library

Nishi Toru, Budde Raymond J A, McMurray John S., Obeyesekere Nihal U., Safdar Naueen, Levin Victor A., Saya Hideyuki

FEBS Letters 399 ( 3 ) 237 - 240 1996.12

ISSN 0014-5793

A bacteriophage peptide library containing a random 15-amino-acid insert was screened for identification of peptide sequence(s) that bind pp60(c-src). Sequencing the random insert from more than 100 virions indicated that more than 60% of the phage virions that bound to this enzyme contained a GXXG sequence motif in which X was frequently a hydrophobic residue. The GXXG sequence was often repeated as GXXGXXG. Two nonameric peptides were synthesized to determine whether or not the peptide inhibits pp60(c-src) tyrosine kinase activity and the importance of the glycine residues within this sequence. The peptide containing glycine had a K(i) of 24 μM, whereas replacing the glycines with proline increased the K(i) value to 3.1 mM.

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Receptor protein tyrosine kinase DDR is up-regulated by p53 protein

Sakuma Shirou, Saya Hideyuki, Tada Mitsuhiro, Nakao Mitsuyoshi, Fujiwara Toshiyoshi, Roth Jack A., Sawamura Yutaka, Shinohe Yumiko, Hiroshi Abe

FEBS Letters 398 ( 2-3 ) 165 - 169 1996.12

ISSN 0014-5793

We have previously reported on radiation-induction of ptk-3 in rat astr ocyte culture [Sakuma et al. (1995) Radiat. Res. 143, 1-7]. Ptk-3 was considered to be a rat version of human DDR (discoidin domain receptor). We cloned and analyzed genomic DNA of the DDR and its promoter region. We discovered that the promoter region contained a consensus sequence of the p53 tumor suppressor binding site. Adenovirus-mediated p53 transfection induced a high level of DDR mRNA in SAOS2 human osteosarcoma cells. These results indicate that DDR is up-regulated by the p53 protein.

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Evidence for p53-mediated modulation of neuronal viability

Xiang Hong, Hochman Daryl W., Saya Hideyuki, Fujiwara Toshiyoshi, Schwartzkroin Philip A., Morrison Richard S.

Journal of Neuroscience 16 ( 21 ) 6753 - 6765 1996.11

ISSN 0270-6474

A role for p53-related modulation of neuronal viability has been suggested by the finding that p53 expression is increased in damaged neurons in models of ischemia and epilepsy. These findings were recently extended with the demonstration that mice deficient in p53 ('knock-out' mice) exhibit almost complete protection from seizure-induced brain injury, whereas wild- type mice display significant neuronal cell loss in the hippocampus and other brain regions. Because the p53 knockout mice used in the latter study expressed a global p53 deficiency in all cell types, it was not possible to conclude that protection was conferred by the exclusive absence of p53 in neurons. Therefore, in the present study, we determined whether p53 expression in isolated neurons is directly coupled to a loss of viability associated with excitotoxic challenge. Primary cultures of hippocampal or cortical neurons were derived from animals containing p53 (+/+, +/-) or those deficient in p53 (-/-). p53-Deficient neurons appeared identical to wild- type neurons with respect to morphology, neurofilament expression, and resting levels of intracellular calcium. Neurons containing at least one copy of p53 were severely damaged by exposure to kainic acid or glutamate. Cell damage was assessed by direct cell counting and by nuclear morphology after propidium iodide staining of DNA. In contrast, neurons deficient in p53 (-/- ) exhibited little or no damage in response to excitotoxin treatment. Despite their divergent outcomes, p53 (+/+) and p53 (-/-) neurons demonstrated similar sustained elevations in intracellular calcium levels triggered by glutamate exposure. Restoring p53 expression to p53-deficient neurons, using adenovirus-mediated transduction, was sufficient to promote neuronal cell death even in the absence of excitotoxin. These results demonstrate a direct relationship between p53 expression and loss of viability in CNS neurons.

Phosphorylation of neurofibromatosis type 1 gene product (neurofibromin) by cAMP-dependent protein kinase

Izawa Ichiro, Tamaki Norihiko, Saya Hideyuki

FEBS Letters 382 ( 1-2 ) 53 - 59 1996.03

ISSN 0014-5793

The critical function of the neurofibromatosis type 1 (NF1) gene product (neurofibromin) is not well defined except that neurofibromin has homology with a family of the GTPase-activating proteins (GAPs). In this study, we confirmed that neurofibromin is constitutively phosphorylated and detected kinase activities which specifically phosphorylate the cystein/serine-rich domain and the C-terminal domain of the neurofibromin in cell lysate. In vitro and in-gel kinase assays strongly indicated that cAMP-dependent protein kinase (PKA) is a candidate for the neurofibromin kinase. The biological significance of the phosphorylation of neurofibromin is unclear at present, but we speculate that neurofibromin plays a crucial role in cellular function since it links the two major cellular pathways which are the GAP-ras and PKA-associated signals.

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High-efficiency in vivo gene transfer using intraarterial plasmid DNA injection following in vivo electroporation

Nishi Toru, Yoshizato Kimio, Yamashiro Shigeo, Takeshima Hideo, Sato Kyoichi, Hamada Kazuya, Kitamura Isao, Yoshimura Teizo, Saya Hideyuki, Kuratsu Jun Ichi, Ushio Yukitaka

Cancer Research 56 ( 5 ) 1050 - 1055 1996.03

ISSN 0008-5472

A novel method for high-efficiency and region-controlled in vivo gene transfer was developed by combining in vivo electroporation and intraarterial plasmid DNA injection. A mammalian expression plasmid for the Escherichia coli lacZ gene (driven with a SV40 early promoter) was injected into the internal carotid artery of rats whose brain tumors (from prior inoculation) had been electroporated between two electrodes. The lacZ gene was efficiently transferred and expressed in the tumor cells 3 days after plasmid injection. However, neither any gene transfers nor any elevated lacZ activity was detected in tissues outside the electrodes. The plasmid was not transferred without electroporation. Human monocyte chemoattractant protein-1 cDNA was also transferred by this method, and its long-lasting (3 weeks) expression was confirmed by using the Epstein-Barr virus episomal replicon system. The expressed monocyte chemoattractant protein-1 protein was functional, as evident by the presence of a large number of monocytes in the tumor tissue. This method, 'electrogene therapy,' which does not require viral genes or particles, allows genes to be transferred and expressed in desired organs or tissues, and it may lead to the development of a new type of highly effective gene therapy.

Transfection of wild-type TP53 induces differentiation in human gingival carcinoma cells

Takahashi R., Inagaki T., Matsuwari S., Fujioka M., Maeda S., Ijuhin N., Haga H., Koh T., Shimada K., Saya H.

European Journal of Cancer 32 ( 3 ) 533 - 539 1996.03

ISSN 0959-8049

We investigated the effects of transfection of wild-type TP53 on the growth properties of a human gingival carcinoma cell line, KOSC-3, in which the TP53 gene is mutated at codon 248 and overexpressed. The wild-type TP53 expression plasmid, pCDM8-p53/neo and the control plasmid, pCDM8/neo, were each stably transfected into KOSC-3 cells by using the calcium phosphate method. The number of G418-resistant colonies from wild-type TP53-transfected cells was approximately half that from plasmid controls. Exogenous wild-type TP53 transcripts were identified in four of the 20 G418-resistant clones analysed by reverse transcription PCR. Although the growth rates of the wild-type TP53+ clones did not drastically change during log phase, their saturation density was significantly reduced. The wild-type TP53+ cells were morphologically flat and enlarged when cultured in vitro, and were less able to form colonies in soft agar. In nude mice, the wild-type TP53+ clones formed subcutaneous tumours with conspicuous keratinisation and notable cell death that was not manifested in the parental and plasmid control cells. These findings indicate that the wild-type TP53 gene, even when it coexists with a mutated form, may function as a growth suppressor and differentiation inducer under restricted conditions in gingival squamous cell carcinoma.

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Oncogenes and tumor suppressor genes

Saya Hideyuki

Japanese Journal of Human Genetics 41 ( 1 ) 18 1996

ISSN 0916-8478

Proliferation and differentiation of cells are known to be regulated by complicated interactions in which various intracellular molecules are involved. Recent molecular genetic analyses have revealed that the genesis and progression of tumors are the result of accumulated changes in two major classes of growthregulatory genes - the oncogenes and the tumor suppressor genes. Several sequential genetic alterations, especially the inactivation of tumor suppressor genes, appear to allow cells to develop toward a more malignant phenotype. More than 10 tumor suppressor genes have been identified since the retinoblastoma susceptibility gene (RB1) was found as the first suppressor gene of mammalian cells in 1986. These genes can be grouped into four classes according to the functions; genes which are involved in 1) regulation of cell cycle, 2) DNA mismatch repair pathway, 3) cell-cell contact signals, and 4) regulation of ras proto-oncogene product. Especially, genes which encode cell cycle regulatory proteins such as RB1, p53, and p!6 have been found to have frequent mutations in various cancer cells, and these functions are rapidly being characterized. The biochemical characters of these cell cycle regulators will be discussed.

Imprinted regulation in the region of Prader-Willi and Angelman syndromes

Nakao Mitsuyoshi, Anan Tadashi, Saya Hideyuki, Sutcliffe James S., Gunaratne Preethi H., Beaudet Arthur L.

Japanese Journal of Human Genetics 41 ( 1 ) 84 1996

ISSN 0916-8478

Prader-Willi syndrome(PWS) and Angelman syndrome(AS) are distinct clinical phenotypes resulting from paternal and maternal deficiencies respectively in human chromosome 15qll-ql3, and are caused by deletion, uniparental disomy, or other mutations. Three paternally expressed transcripts containing small nuclear ribonucleoproteinassociated polypeptide N(SNRPN), PAR-5 and PAR-1 were absent in cultured cells from PWS patients with a small deletion that involve a differentially methylated CpG island around a 5' untranslated exon alpha of SNRPN gene. Methylation of the CpG island is specific for the maternal chromosome consistent with paternal expression of the imprinted domain. The small deletion, which is benign when maternally transmitted, extends upstream 50 kb from the CpG island, and results in the loss of expression of the three imprinted transcripts, altered methylation and replication timing around SNRPN gene, implying the presence of an imprinting control element.

Microsatellite analysis of chromosome 10q in malignant glioma

Nakamura Hideo, Nakao Mitsuvoshi, Saya Hideyuki, Kuratsu Jun Ichi, Ushio Yukitaka

Japanese Journal of Human Genetics 41 ( 1 ) 57 1996

ISSN 0916-8478

Chromosome 10 is frequently deleted in malignant gliomas and the frequency of the deletion is known to be related to clinical and pathological malignancy of the tumor. Therefore, we expect that certain genes which is implicated in malignancy of glioma are located on chromosome 10. Here, we analyze the loss of heterozygosity (LOH) of microsatellite markers on chromosome lOq by PCR using fluorescentry-labelled primer. D10S201, D10S200, D10S603, D10S192, D10S205, D10S222, D10S540, D10S566, D10S530, D10S597, D10S545, D10S587, D10S186, and D10S212 were used for this analysis. LOH of the markers were quantitatively analyzed by an automated DNA fragment analyzer and a software. Using this system, we found LOH in 19 of 43 cases of malignant gliomas, and the most frequent deletion was identified at D10S 566 region.

CD44 hyaluronate binding influences growth kinetics and tumorigenicity of human colon carcinomas

Takahashi K., Stamenkovic I., Cutler M., Saya H., Tanabe K. K.

Oncogene 11 ( 11 ) 2222 - 2232 1995.12

ISSN 0950-9232

CD44 is a cell surface receptor for hyaluronate that is implicated in the regulation of tumor growth and metastatic potential. Transformation of colon mucosa to carcinoma is associated with overexpression of several CD44 alternative splice variants. The functional roles of CD44 isoforms in colon carcinoma tumor progression remain unclear. CD44H expression is downregulated in colon carcinomas compared to paired normal mucosa. In the present study we demonstrate that reintroduction of CD44H back into colon carcinoma cells by stable transfection reduces their in vitro growth rate and tumorigenicity. Examination of several colon carcinoma cell lines and use of mutant CD44H reveal that this in vitro and in vivo growth reduction requires the ability of CD44H to bind hyaluronate. These observations indicate that the CD44H downregulation associated with transformation of mucosa to colon carcinoma may provide the carcinoma cells with a growth advantage. Furthermore, the reduction in tumor growth rate mediated by reintroduction of CD44H into colon carcinoma cells is dependent on its ability to bind hyaluronate.

Induction of Fas-mediated apoptosis in p53-transfected human colon carcinoma cells

Tamura T., Aoyama N., Saya H., Haga H., Futami S., Miyamoto M., Koh T., Ariyasu T., Tachi M., Kasuga M., Takahashi R.

Oncogene 11 ( 10 ) 1938 - 1946 1995.11

ISSN 0950-9232

To investigate the biological function of p53 in colon carcinoma cells, a wild-type p53 expression plasmid under the control of the human cytomegalovirus promoter was stably transfected into the human colon adenocarcinoma cell line WiDr, which carries a mutation of the p53 gene at codon 273. Exogenous wild-type p53 transcripts were detected at various expression levels in 8 of 117 G418-resistant clones. The growth rates of the wild-type p53+ clones in culture did not change significantly. The efficiency of colony formation in soft agar, however, was completely suppressed in two wild-type p53+ clones. This is the first to demonstrate the feasibility of stable transfection of the wild-type p53 gene under the control of non-inducible promoter in human colon cancer cells. The major alteration found was that wild-type p53+ cells which were incubated with anti-Fas IgM showed marked cytolysis with preferential overexpression of wild-type p53 accompanied by overexpression of a cyclin-dependent kinase inhibitor, WAF1, whereas the endogenous mutant p53 retained its expression level. The findings suggest that a Fas-initiated pathway is incidentally linked to a p53-dependent apoptotic pathway through the reconstituted wild-type p53 gene in WiDr cells. This model should help elucidating the additional role of the p53 tumor suppressor gene and the mechanism of apoptosis in colon carcinoma cells.

Implication of cell cycle regulating genes in carcinogenesis

Hanada N., Saya H.

Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 40 ( 11 ) 1713 - 1720 1995.08

ISSN 0039-9450

Discovery, development, and testing of substrates and inhibitors of pp60^c-SRC

Budde Raymond J A, Mcmurray John S., Saya Hideyuki, Gallick Gary E., Levin Victor A.

Pharmaceutical Biology 33 ( S1 ) 27 - 34 1995

ISSN 1388-0209

Currently, there are no specific protein tyrosine kinase inhibitors available. This review summarizes our efforts to develop an active-site-directed inhibitor of pp60^c-srv Initial efforts are directed at determining substrate specificity with synthetic peptides and at developing a biological system to test the potential of pp60^c-src inhibitors to effectively inhibit the growth of pp60^c-src activated cell lines. To meet these goals, we have developed new methods for purifying recombinant pp60^C-SRC, assaying tyrosine kinase activity, synthesizing cyclic peptides, and generating random peptide libraries. In addition, we discuss the generation of potential artifacts while using polyhydroxy aromatic compounds as tyrosine kinase inhibitors.

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Discovery, development, and testing of substrates and inhibitors of pp60(c-src)

Budde R. J A, McMurray J. S., Saya H., Gallick G. E., Levin V. A.

International Journal of Pharmacognosy 33 ( SUPPL. ) 27 - 34 1995

ISSN 0925-1618

Currently, there are no specific protein tyrosine kinase inhibitors available. This review summarizes our efforts to develop an active-site-directed inhibitor of pp60(c-src). Initial efforts are directed at determining substrate specificity with synthetic peptides and at developing a biological system to test the potential of pp60(c-src) inhibitors to effectively inhibit the growth of pp60(c-src) activated cell lines. To meet these goals, we have developed new methods for purifying recombinant pp60(c-src), assaying tyrosine kinase activity, synthesizing cyclic peptides, and generating random peptide libraries. In addition, we discuss the generation of potential artifacts while using polyhydroxy aromatic compounds as tyrosine kinase inhibitors.

Alternative RNA splicing of the hyaluronic acid receptor CD44 in the normal human brain and in brain tumors

Nagasaka S., Tanabe K. K., Bruner J. M., Saya H., Sawaya R. E., Morrison R. S.

Journal of Neurosurgery 82 ( 5 ) 858 - 863 1995

ISSN 0022-3085

The cell-surface receptor for hyaluronic acid, CD44, is expressed by both normal and malignant cells. Numerous CD44 isoforms have recently been identified that are derived by alternative ribonucleic acid splicing. The expression of some CD44 isoforms has been shown to be involved in tumor progression and metastatic spread in a rat carcinoma model and in human carcinomas. In the present study, CD44 isoform expression was evaluated by reverse transcriptase-polymerase chain reaction (PCR) analysis in frozen sections derived from three samples of normal brain tissue and from 40 brain tumors, including samples of glioblastoma multiforme, anaplastic astrocytoma, low-grade astrocytoma, cerebral primitive neuroectodermal tumor, medulloblastoma, metastatic colon carcinoma, and metastatic melanoma. Normal brain tissue adjacent to the tumors was also examined in 14 of 18 glioblastomas. In all normal brain and tumor samples, with the exception of metastases from colon carcinoma, PCR analysis demonstrated one prominent product that corresponded to the CD44H hematopoietic form of CD44. Metastases from colon carcinoma demonstrated two prominent PCR amplification products corresponding to CD44H and CD44R1. These results suggest that CD44H is the predominant isoform of this protein in normal human brain tissue and in human neuroectodermal tumors of varying degrees of malignancy. The ability of CD44H to mediate tumor cell motility and invasiveness (in contrast to CD44R1) suggests that the CD44 alternative splicing pattern of neuroectoderm-derived tumors may enhance their local biological aggressiveness and intracerebral spread. The lack of expression of larger molecular weight CD44 variants by primary brain tumors may also partially explain why these tumors rarely metastasize to distant sites.

Radiation induction of the receptor tyrosine kinase gene Ptk-3 in normal rat astrocytes

Sakuma S., Saya H., Ijichi A., Tofilon P. J.

Radiation Research 143 ( 1 ) 1 - 7 1995

ISSN 0033-7587

Radiation-induced gene expression was examined in rat astrocyte cultures using differential display of mRNA via reverse transcriptase-polymerase chain reaction. A 0.3-kb cDNA that was consistently observed in irradiated cultures but not in unirradiated cultures was cloned and sequenced. It was found to be identical to Ptk-3, a receptor tyrosine kinase gene identified recently. The protein encoded by Ptk-3 is a member of a novel class of receptor tyrosine kinases whose extracellular domain contains regions of homology with coagulation factors V and VIII and complement component C1. Northern blot analysis revealed that the expression of Ptk-3 was increased in rat astrocytes by 0.5 h after exposure to 10 Gy and remained at the same elevated level for at least 24 h. The maximum increase occurred after 5 Gy. Cloning studies indicated the presence of at least two Ptk-3 mRNA transcripts, which are probably the result of an alternative splicing mechanism. The short isoform lacks a 37-amino acid sequence in the glycine/proline-rich juxtamembrane region. The splicing pattern of the Ptk-3 gene was not altered by radiation. However, the ratios of the longer to the shorter mRNA transcripts differed between adult cortex, neonatal cortex and in vitro astrocyte cultures.

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Basic fibroblast growth factor and fibroblast growth factor receptor I are implicated in the growth of human astrocytomas

Morrison Richard S., Yamaguchi Fumio, Saya Hideyuki, Bruner Janet M., Yahanda Alan M., Donehower Lawrence A., Berger Mitchel

Journal of Neuro-Oncology 18 ( 3 ) 207 - 216 1994.10

ISSN 0167-594X

Malignant astrocytomas are highly invasive, vascular neoplasms that comprise the majority of nervous system tumors in humans. A strong association has previously been made between malignancy in human astrocytic tumors and increased expression of certain fibroblast growth factor (FGF) family members, including basic and acidic FGF. The influence of endogenous basic FGF on glioblastoma cell growth in vitro was evaluated using basic FGF-specific antisense oligonucleotides. These studies indicated that human glioblastoma cell growth in vitro, can be inhibited by suppressing basic FGF expression. Human astrocytomas also exhibited changes in FGF receptor (FGFR) expression during the course of their progression from a benign to a malignant phenotype. FGFR2 (bek) expression was abundant in normal white matter and in all low grade astrocytomas, but was not observed in glioblastomas. Conversely, FGFR1 (flg) expression was absent or barely detectable in normal white matter, but was significantly elevated in glioblastomas. Glioblastomas also expressed an alternatively spliced form of FGFR1 containing two immunoglobulin-like disulfide loops (FGFR1β), whereas normal human adult and fetal brain expressed a form of the receptor containing three immunoglobulin-like disulfide loops (FGFR1α). Intermediate grades of astrocytic tumors exhibited a gradual loss of FGFR2 and a shift in expression from FGFR1α to FGFR1β as they progressed from a benigh to a malignant phenotype. The underlying cytogenetic changes that contribute to these alterations are not entirely understood, but abnormalities in the p53 tumor suppressor gene may influence expression of bFGF as well as the FGFR. These results suggest that alterations in FGFR signal transduction pathways may play a critical role in the malignant progression of astrocytic tumors.

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Detection of cellular proteins that interact with the NF2 tumor suppressor gene product

Takeshima Hideo, Izawa Ichiro, Lee Polly S Y, Safdar Naueen, Levin Victor A., Saya Hideyuki

Oncogene 9 ( 8 ) 2135 - 2144 1994.08

ISSN 0950-9232

The neurofibromatosis type 2 (NF2) gene was recently cloned, and the protein it encodes (merlin) was revealed to belong to a family of proteins that link cytoskeletal components with proteins in the cell membrane. To elucidate the biological function of merlin, we produced a bacterial fusion protein consisting of glutathione S-transferase and merlin and used it to detect five merlin-binding cellular proteins, designated p165, p145, p125, p85 and p70, by a protein-binding assay. p165 and merlin were phosphorylated on serine/threonine residues, and immunoprecipitation showed that p85 bound the native form of merlin. Although the entire merlin-ezrin-radixin-moesin (MERM) homology domain of merlin was found to be essential for binding to all five proteins, the MERM homology domains of ezrin and moesin did not bind to any of the five proteins. Since most reported NF2 mutations are in the region we determined was necessary for binding, the mutations probably impair binding. Therefore, the formation of the protein complex is probably crucial for tumor suppression.

Germline p53 gene mutations in subsets of glioma patients

Kyritsis Athanassios P., Bondy Melissa L., Xiao Mi, Berman Elise L., Cunningham Joan E., Lee Polly S., Levin Victor A., Saya Hideyuki

Journal of the National Cancer Institute 86 ( 5 ) 344 - 349 1994.03

ISSN 0027-8874

Background: Heritable germline mutations of the p53 gene have been described in patients with Li-Fraumeni syndrome, occasionally in nonfamilial malignancies such as multifocal osteosarcoma, in a small subgroup of young patients with two or more primary malignancies, and in patients with sporadic breast carcinoma. We recently reported that multifocal gliomas are frequently associated with other primary malignancies, and we hypothesized that genetic alterations may account for this phenomenon. Purpose: We examined the frequency of germline p53 gene mutations in patients with glioma and either multifocality of lesions, history of an additional primary (different) malignancy, or a family history of cancer. Methods: Lymphocytes from 51 glioma patients were analyzed for germline p53 gene mutations using RNA- polymerase chain reaction analysis, single-strand conformation polymorphism, and gene sequencing techniques. Results: Germline p53 gene mutations were detected in six of 19 patients with multifocal glioma, including two with family history of cancer, one with another primary malignancy, and two with all three risk factors; one of four patients with unifocal glioma, another primary malignancy, and a family history of cancer; and two of 15 patients with unifocal glioma and a family history of cancer but no second malignancies. No mutations were detected in the patient with unifocal glioma and another malignancy or in the 12 control patients with unifocal glioma and no second malignancies or family history of cancer. Patients having mutations were younger than other patients in the same group. Conclusions: Germline p53 mutations are frequent in patients with multifocal glioma, glioma and another primary malignancy, and glioma associated with a family history of cancer, particularly if these factors are combined. Implications: Relatives at high risk can be identified for genetic counseling, early cancer detection, and possible enrollment in chemoprevention trials.

Differential expression of two fibroblast growth factor-receptor genes is associated with malignant progression in human astrocytomas

Yamaguchi Fumio, Saya Hideyuki, Bruner Janet M., Morrison Richard S.

Proceedings of the National Academy of Sciences of the United States of America 91 ( 2 ) 484 - 488 1994.01

ISSN 0027-8424

Malignant astrocytomas, which are highly invasive, vascular neoplasms, compose the majority of nervous system tumors in humans. Elevated expression of fibroblast growth factors (FGFs) in astrocytomas has implicated the FGF family of mitogens in the initiation and progression of astrocyte-derived tumors. In this study, we demonstrated that human astrocytomas undergo parallel changes in FGF-receptor (FGFR) expression during their progression from a benign to a malignant phenotype. FGFR type 2 (BEK) expression was abundant in normal white matter and in all low-grade astrocytomas but was not seen in malignant astrocytomas. Conversely, FGFR type 1 (FLG) expression was absent or barely detectable in normal white matter but was significantly elevated in malignant astrocytomas. Malignant astrocytomas also expressed an alternatively spliced form of FGFR-1 (FGFR-1β) containing two immunoglobulin-like disulfide loops, whereas normal human adult and fetal brains expressed a receptor form (FGFR-1α) containing three immunoglobulin- like disulfide loops. Intermediate grades of astrocytic tumors exhibited a gradual loss of FGFR-2 and a shift in expression from FGFR-1α to FGFR-1β as they progressed from benign to malignant phenotype. These results suggest that differential expression and alternative splicing of FGFRs may be critical in the malignant progression of astrocytic tumors.

The CD44 adhesion molecule and metastasis

Tanabe K. K., Saya H.

Critical Reviews in Oncogenesis 5 ( 2-3 ) 201 - 212 1994

ISSN 0893-9675

Polyclonal uses of T-cell receptor (TCR)α and β genes for cytotoxic T lymphocytes in human metastatic melanoma

Seito D., Morita T., Masuoka K., Maeda T., Saya H., Itoh K.

International Journal of Cancer 58 ( 4 ) 497 - 502 1994

ISSN 0020-7136

Identification of genetic structure and diversity of T-cell receptor (TCR)α and β genes for cytotoxic T lymphocytes (CTLs) infiltrating human cancers is important for the better understanding of molecular mechanisms of host defense at tumor sites. cDNAs of TCRα and β genes of 22 different melanoma-specific CTL clones established from the tumor-infiltrating lymphocytes of 2 patients were sequenced for analysis of their genetic structure and diversity. Vα7.2-Jα10-Cα was found in 4 of 22 clones, 2 of which also used the same β-chain. The other 20 clones showed different combinations of α and β use. At deduced amino-acid levels, 7 of 9 clones from one patient used a threonine residue at the 26th position in the complementarity-determining region (CDR) 1 of TCRα. Eight of 13 clones used a threonine at the 99th or a serine residue at the 100th position in CDR3 of TCRα. CTL clones with the same or different TCRαα showed the same or different patterns of cytotoxicity, respectively. These results suggest that CTLs usually do not demonstrate clonal expansion at tumor sites of metastatic melanoma's but rather that polyclonal T cells capable of binding to multiple melanoma determinants through CDR3 of TCRα accumulate in the tumor.

Increase in the capability of interleukin 2 gene-transduced renal cell carcinoma cells to induce cytotoxic lymphocytes.

Itoh K., Umezu Y., Morita T., Saya H., Seito D., Augustus L. B., Nakao M., Sakata M., Miyajima J., Masuoka K.

Kurume Medical Journal 41 ( 2 ) 53 - 63 1994

ISSN 0023-5679

The immunogenicity of human cancer cells transfected with interleukin 2 (IL-2) gene, a potent vaccine candidate, has not yet been fully investigated. Human renal cell carcinoma (RCC) cells transduced with human IL-2 gene (RCC-IL-2) were investigated in vitro for the capability to induce lymphokine-activated killer (LAK) or cytotoxic T lymphocyte (CTL) activity in peripheral blood mononuclear cells (PBMC) or tumor-infiltrating lymphocyte (TIL). The RCC-IL-2 cells stimulated PBMC to demonstrate LAK activity, and also stimulated autologous TILs to proliferate and exhibit cytotoxicity relatively restricted to autologous tumor cells. In contrast, both parental RCC and RCC transduced with neomycin gene alone failed to induce these activities. These results indicate that RCC-IL-2 cells are more potent than the other RCC cells with regard to inducing cytotoxic lymphocytes against autologous tumor cells.

Bacterial expression of an active tyrosine kinase from a protein A/truncated c-src fusion protein

Saya Hideyuki, Lee Polly S Y, Nishi Toru, Izawa Ichiro, Nakajima Motowo, Gallick Gary E., Levin Victor A.

FEBS Letters 327 ( 2 ) 224 - 230 1993.07

ISSN 0014-5793

The carboxy-terminal half of the c-src protein fused to the protein A moiety was expressed in bacteria. The protein A/truncated c-src fusion protein, which does not have SH2 and SH3 domains, is found in the periplasmic space allowing for a simple one-step purification and demonstrated high efficiency in autophosphorylation and exogeneous substrate phosphorylation. The missense mutation at codon 294 (Ile → Thr), which is located in the ATP-binding domain of the c-src, resulted in dramatic reduction of tyrosine kinase activity of the fusion protein. Using the fusion protein. we also revealed that staurosporin, a well-known kinase inhibitor, directly affects autophosphorylation of the C-terminal half of the c-src protein. This truncated c-src expression system provides a good source of enzyme for diverse experiments and is an ideal model for understanding the implication of structural alterations in the catalytic activity of the c-src kinase by site-directed mutagenesis experiments.

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Detection of p53 alterations in human astrocytomas using frozen tissue sections for the polymerase chain reaction

Aka Koji, Bruner Janet M., Bondy Melissa L., Ligon Keith, Nishi Toru, del Giglio Auro, Moser Richard P., Levin Victor A., Saya Hideyuki

Journal of Neuro-Oncology 16 ( 2 ) 125 - 133 1993.06

ISSN 0167-594X

The polymorphism of amino acid residue 72 on the human p53 tumor-suppressor gene is a useful marker for detecting intragenic loss of heterozygosity (LOH). We examined the LOH of the p53 gene in human malignant astrocytomas by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis using DNA extracted from frozen tissue sections under histologic examination. Eleven of 16 informative cases (69%) of the malignant astrocytomas were found to have LOH in the p53 gene. Sequential frozen sections were analyzed by immunohistochemistry using anti-p53 antibody PAb1801 to detect overexpression of the p53 protein, which is presumably altered if it is detectable. Ten of the 11 cases that had LOH of the p53 gene overexpressed the p53 protein. Moreover, 4 of the 11 patients with LOH of the p53 gene developed a second neoplasm in addition to an astrocytoma, possibly indicating genetic instability in these patients. These data suggest that alterations of the p53 gene may play an important role in the genesis of malignant astrocytoma. The combination of the PCR-RFLP method and immunohistochemical analysis using frozen tissue sections is a practical diagnostic tool for examination of human malignancies, including astrocytomas.

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Expression of CD44R1 adhesion molecule in colon carcinomas and metastases

Tanabe K. K., Ellis L. M., Saya H.

The Lancet 341 ( 8847 ) 725 - 726 1993.03

ISSN 0140-6736

CD44 is a cell surface adhesion molecule postulated to control lymphocyte recirculation by facilitating entry into lymphoid tissue. Tumour cells transfected to overexpress the epithelial variant CD44R1 readily gain access to lymph nodes and distant metastatic sites in animal models, possibly by mimicking circulating lymphocytes.. To investigate if human tumours display altered CD44 expression we performed reverse transcriptase a polymerase chain reaction (PCR) analysis of CD44 in 49 specimens from normal colonic mucosa, primary colon and rectal tumours, normal liver, and metastases of 20 patients. The haematopoietic variant CD44H was the principal isoform amplified in all of the specimens, However, 12/14 primary tumours and 16/16 metastatic tumours expressed CD44R1, compared with only 2 of 13 normal mucosa specimens. This increase in CD44R1 relative to CD44H expressed by human colon carcinoma cells may increase their metastatic potential. Moreover, the relative increase in PCR amplification of CD44R1 compared with that of CD44H may provide a sensitive method for detecting primary and metastatic colon carcinoma cells in small biopsy specimens.

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p53 protein immunostaining in routinely processed paraffin-embedded sections.

Bruner J. M., Connelly J. H., Saya H.

Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 6 ( 2 ) 189 - 194 1993.03

ISSN 0893-3952

Mutation of the p53 tumor suppressor gene is the most common genetic alteration in human tumors. The altered protein product of the mutant gene is stabilized in tumor cells and can be detected using monoclonal antibody immunohistochemistry. We report a technique for immunostaining of the altered p53 protein in routinely processed paraffin-embedded tissue sections, comparing this method with a frozen-section immunostain method for concordance. Similarly processed tumor cell lines with known mutations or deletions of the p53 gene served as positive or negative controls. The results in 25 human gliomas and 20 colorectal carcinomas showed a 100% correspondence of positive reactivity in the colorectal carcinomas and an 83% correspondence in the gliomas. In no case did the paraffin-embedded sections react in the absence of frozen section reactivity (that is, there were no false positives). Three of 18 gliomas showed reactivity in frozen sections without reactivity in the companion paraffin-embedded sections. This discrepancy could have been caused by technical factors such as length of fixation time. We propose that this new paraffin-embedded section immunostaining method will be of value as a screening technique for the investigation of p53 mutations in archived human tumors. The data thus obtained may then be correlated with clinical information and perhaps be of value in diagnosis or predicting outcome for various human cancers.

Molecular pathogenesis and management of gliomas

Kyritsis A. P., Saya H., Levin V. A.

Neuroimaging Clinics of North America 3 ( 4 ) 735 - 744 1993

ISSN 1052-5149

Reduced tumorigenicity and cell motility of a gastric carcinoma cell line by introduction of wild-type p53 gene

Kyo E., Yokozaki H., Yanagihara K., Yasui W., Ito M., Sano T., Lee P. S., Saya H., Tahara E.

International Journal of Oncology 3 ( 2 ) 265 - 271 1993

ISSN 1019-6439

Wild-type or mutant human p53 gene was transfected into a human gastric carcinoma cell line MKN-1 which shares a mutant p53 allele. Transfected wild- type p53 reduced the colony forming efficiency and tumorigenicity of MKN-1 cells. However, no difference in expression of cell adhesion molecule, oncogenes and growth factors was observed among parent, wild-type p53 and mutant p53 transfectants. In motility assay, the wild-type p53 transfectants relative to the parental or mutant p53 transfectants exhibited a decreased motility, and HGF had a greater effect on the motility of the mutant p53 transfectants, but very little effect on the motility of either the parental or wild-type transfectants. In invasion assay, mutant p53 transfectants revealed the increased invasion ability into collagen gel. These results suggest that allele loss and point mutation of p53 gene may play a critical role not only in growth but also in invasion of gastric carcinoma cells.

Proliferating cell nuclear antigen (PCNA) expression in chronic lymphocytic leukemia (CLL)

Giglio Auro Del, O'brien Susan, Ford Richard J., Manning John, Saya Hideyuki, Keating Michael, Johnston Dennis, Chamone Dalton Fisher, Deisseroth Albert B.

Leukemia and Lymphoma 10 ( 4-5 ) 265 - 271 1993

ISSN 1042-8194

Chronic Lymphocytic Leukemia (CLL) is usually an indolent disorder which in some patients assumes an aggressive clinical course. In order to assess at presentation the prognosis of a given patient, several staging systems and prognostic variables have been proposed including the expression of the Proliferating Cell Nuclear Antigen (PCNA). PCNA is a 36 kd nuclear protein, the regulation of which is cell cycle-dependent. In CLL, PCNA levels correlate with cell proliferation, clinical stage and the lymphocyte doubling time (LDT). Furthermore, preliminary data suggests that PCNA expression may also predict response to Fludarabine-based chemotherapy. Since PCNA is a cofactor for Delta DNA polymerase, PCNA overexpression in CLL may also reflect the intrinsic DNA repair activity of the leukemic cells and thus their resistance to chemotherapy. Further studies aiming at modulation of PCNA expression in CLL cells may clarify this issue and may offer a future new therapeutic strategy with which to treat this disorder.

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Role and regulation of expression of 92-kDa type-IV collagenase (MMP-9) in 2 invasive squamous-cell-carcinoma cell lines of the oral cavity

Juarez J., Clayman G., Nakajima M., Tanabe K. K., Saya H., Nicolson G. L., Boyd D.

International Journal of Cancer 55 ( 1 ) 10 - 18 1993

ISSN 0020-7136

The present study was undertaken to determine the role of the metalloproteinase MMP-9 in the invasive phenotype of squamous-cell carcinoma of the oral cavity and the regulation of its expression. Zymographic analysis of conditioned medium from 2 highly invasive squamous-cell-carcinoma cell lines indicated large amounts of an enzyme which was indistinguishable, in size (92 kDa) from the MMP-9 pro-enzyme. Conversion of the 92-kDa gelatinase into a lower-molecular-weight species (84 kDa), identical in size to the activated gelatinase, was evident when both cell lines, which are avid secretors of urokinase, were cultured in the presence of plasminogen. Penetration of an extracellular-matrix-coated filter was dramatically reduced in the presence of the collagenase inhibitor, tissue inhibitor of metalloproteinase-2, suggesting a critical role for MMP-9 in the invasive process. Immunohistochemical studies demonstrating the presence of MMP-9 in tumor cells of resected squamous-cell cancers suggested that secretion of this collagenase by cells in vitro was reflective of the in vivo setting. Since several phorbol-ester response elements are present in the MMP-9 promoter, we determined the role of protein-kinase-C pathways in the regulation of MMP-9 expression in cultured SCC. Treatment of cells with PMA resulted in a more-than-20-fold increase in the level of protein and mRNA. Conversely, culturing of cells in the presence of the protein-kinase-C inhibitor, calphostin-C, led to a dose-dependent decrease in the amount of MMP-9 mRNA and protein, suggesting that the constitutive expression of this collagenase reflects activation of this signal transduction pathway. In summary, our data suggest that, for a sub-population of squamous-cell carcinomas, secreted MMP-9 is an important determinant of the invasive phenotype, and that the expression of this metalloproteinase is regulated by protein-kinase-C pathways.

Novel variants of CD44 arising from alternative splicing

Tanabe K. K., Nishi T., Saya H.

Molecular Carcinogenesis 7 ( 4 ) 212 - 220 1993

ISSN 0899-1987

CD44 is a cell-surface glycoprotein postulated to play a role in a variety of biological processes, including lymphocyte homing and tumor-cell metastasis. Several isoforms of CD44 have been identified in human cells, and the genesis of some of these isoforms has been attributed to alternative splicing. In the study presented here we amplified three novel transcript variants of CD44 from human cell lines using a reverse transcriptase-polymerase chain reaction strategy. Two of the novel isoforms differed from previously described CD44 isoforms as a result of alternative splicing that occurred at previously reported splice junctions. The third novel CD44 isoform was generated from a previously unreported alternative splice junction near the 5' end of the open reading frame. Southern blot analysis of genomic DNA revealed that these novel isoforms and all of the previously described CD44 isoforms arose from alternative splicing. The capability of cells to modify their CD44 alternative splicing pattern was demonstrated in MCF-7 cells, which altered their CD44-isoform expression pattern in response to treatment with hyaluronidase. A better understanding of mechanisms regulating CD44 alternative splicing may provide insights into diverse processes, including tumor-cell metastasis annd lymphocyte homing.

Molecular, genetic, and biologic aspects of primary brain tumors

Steck P. A., Bruner J. M., Pershouse M. A., Hadi A., Conrad C. A., Yung W. K A, Saya H.

Cancer Bulletin 45 ( 4 ) 296 - 303 1993

ISSN 0740-820X

Epidemiology, cytogenetics, and molecular biology of brain tumors

Kyritsis A. P., Saya H.

Current Opinion in Oncology 5 ( 3 ) 474 - 480 1993

ISSN 1040-8746

Prognostic value of proliferating cell nuclear antigen expression in chronic lymphoid leukemia

Del Giglio Auro, O'Brien Susan, Ford Richard, Saya Hideyuki, Manning John, Keating Michael, Johnston Dennis, Khetan Rainer, El-Naggar Adel, Deisseroth Albert

Blood 79 ( 10 ) 2717 - 2720 1992.05

ISSN 0006-4971

Chronic lymphocytic leukemia (CLL) is a usually indolent disease that can assume an aggressive clinical course in some patients. To develop assays that would be predictive of how a particular patient's disease would evolve, we studied the expression of proliferating cell nuclear antigen (PCNA) by Western blotting in 40 patients with CLL. The concentration of PCNA, a cofactor for δDNA-dependent DNA polymerase, is indicative of the proliferative state of the cell. Significantly lower PCNA levels were observed in earlier stage CLL when compared with more advanced disease. The leukemic cell proliferative rate, assessed by lymphocyte doubling time and flow cytometry, also correlated significantly with the level of PCNA expression. These results suggest that a high level of PCNA in the cells of CLL patients at presentation identifies a subgroup of patients whose CLL cells have a higher proliferative activity and who may, therefore, have a potentially shorter survival.

Alternative splicing of neurofibromatosis type 1 gene transcript in malignant brain tumors

Mochizuki H., Nishi T., Bruner J. M., Lee P. S Y, Levin V. A., Saya H.

Molecular Carcinogenesis 6 ( 2 ) 83 - 87 1992

ISSN 0899-1987

The neurofibromatosis type 1 (NF1) gene encodes a 360-residue region showing significant homology to the catalytic domains of both mammalian GTPase-activating protein (GAP) and yeast IRA protein. The product of the GAP-related domain of the NF1 gene (NF1-GRD) has been shown to stimulate ras GTPase and consequently to inactivate ras protein. We previously reported that the NF1-GRD has two types of transcripts, type I and type II, which are generated by an alternative splicing mechanism, and that the differential splicing of the NF1-GRD may be related to differentiation of neuroectodermal cells. Here we examined the differential expression of type I and type II transcripts of NF1-GRD in clinical samples of supratentorial malignant brain tumors by the RNA-polymerase chain reaction (PCR) method using frozen tissue sections. Our observations revealed that normal cerebrum predominantly expressed the type II NF1-GRD transcript, whereas primitive neuroectodermal tumors predominantly expressed the type I transcript. Additionally, although the type I/type II ratio in astrocytomas varied widely among tissue samples, all glioblastomas showed higher type I/type II ratios that adjacent brain samples. The RNA-PCR analysis using frozen tissue sections is a useful and sensitive method for detecting genetic markers in clinical tissue samples.

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Structural alterations of the p53 gene in human leukemias

Miwa Hiroshi, Kita Kenkichi, Saya Hideyuki, Otsuji Akira, Masuya Masahiro, Nishii Kazuhiro, Morita Norihisa, Takakura Nobuyuki, Ohishi Koushi, Nasu Kaori, Nakase Kazunori, Shirakawa Shigeru

Leukemia Research 16 ( 11 ) 1105 - 1112 1992

ISSN 0145-2126

We have investigated alterations of the p53 gene in human leukemias by polymerase chain reaction-mediated restriction fragment length polymorphism analysis. This method detects the codon 72 polymorphism of the p53 gene, allowing identification of loss of heterozygosity (LOH) of the p53 gene. In this study, at least two specimens were obtained from each patient to compare the allele status at different points of clinical course. Of 21 cases examined 7 were heterozygous at this polymorphic site and were evaluable for LOH study. Only one patient of Philadelphia chromosomepositive acute lymphoblastic leukemia (ALL) lost the heterozygosity in the specimen of her last relapse. Leukemic cells from her repeated relapses including the last one revealed to have the clonally rearranged immunoglobulin H chain gene of the same size, indicating that alteration of the p53 gene in this patient might account for the lethal relapse as a clonal evolution event. Northern-blot analysis of the p53 gene showed that one Gase of CD7(+) ALL had altered p53 mRNA. Overall, it is demonstrated that alterations of the p53 gene might be involved in the pathogenesis or progression of at least some human leukemias, although the alterations in leukemias seemed to be not as frequent as in solid tumors.

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Neurofibromatosis type 1 (NF1) gene

Nishi Toru, Saya Hideyuki

Cancer and Metastasis Reviews 10 ( 4 ) 301 - 310 1991.12

ISSN 0167-7659

The gene responsible for neurofibromatosis type 1 (NF1), a common autosomal dominantly inherited disease, has been isolated. A region of NF1 gene product has been demonstrated to share structural and functional similarities with the mammalian GTPase activating protein (GAP) and the yeast IRA proteins. Thus, the NF1 protein is thought to play a role in signal transduction by stimulating the conversion of the Ras protein from a GTP-bound active form to a GDP-bound inactive form. The increased risk of malignant tumors in neuroectodermal tissues of NF1 patients may be caused by disruption of growth and differentiation regulatory functions of the NF1 gene. A second type of the NF1-GAP related domain (NF1-GRD) transcript, which has an extra 21-amino-acid insert in the center of the previously reported first type transcript, has been described. This insert significantly changes the hydrophilicity and secondary structure of the central region of NF1-GRD, therefore, suggesting it also changes its function. Alternative splicing is the most likely mechanism by which these two types of transcripts arise. The NF1-GRD alternative splicing has been shown to be intimately involved in differentiation of neuroectodermal tissues. Aberrant regulation of the alternative splicing may contribute to tumor formation in neuroectodermal tissue.

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Effect of herbimycin a on growth and pp60^c-src activity in human colon tumor cell lines

Garcia R., Parikh N. U., Saya H., Gallick G. E.

Oncogene 6 ( 11 ) 1983 - 1989 1991.11

ISSN 0950-9232

The effect of herbimycin A, an ansamycin antibiotic which inhibits cellular transformation by retroviral tyrosine kinases, on the monolayer growth of seven colon tumor cell lines and one cell line established from normal colonic mucosa, CCL239, was examined. Each colon tumor cell line tested showed dose-dependent growth inhibition in response to herbimycin A. A 125 ng ml^-1 dose of the antibiotic caused > 40% growth inhibition in all colon tumor cell lines after two cell doublings. In contrast, at similar herbimycin A concentrations only 12% inhibition was observed in 'normal' CCL239 cells. No major morphologic changes were observed at the light microscopic level in any of the tumor cell lines or CCL239 cells in response to treatment with herbimycin A. Studies using the HT29 colon adenocarcinoma cell line showed dosedependent inactivation of pp60^c-src by herbimycin A, resulting in decreased autophosphorylation, enolase phosphorylation and steady-state levels, which correlated with cellular growth inhibition. Herbimycin A-induced reductions in pp60^c-src kinase activity preceded changes in pp60^c-src steady-state levels. Growth and pp60^c-src inhibition were reversible following removal of herbimycin A from cell culture media. Our results suggest that regulation of pp60^c-src tyrosine kinase activity may be important in growth control of colon tumor cells.

Differential expression of two types of the neurofibromatosis type 1 (NF1) gene transcripts related to neuronal differentiation

Nishi Toru, Lee Polly S Y, Oka Koji, Levin Victor A., Tanase Sumio, Morino Yoshimasa, Saya Hideyuki

Oncogene 6 ( 9 ) 1555 - 1559 1991.09

ISSN 0950-9232

A 360 residue region encoded by the neurofibromatosis type 1 (NF1) gene shows significant homology to the catalytic domains of both mammalian GTPase-activating proteins (GAP) and yeast IRA proteins. This GAP-related domain of the NF1 gene (NF1-GRD), like the GAP and IRA protein, has been reported to mediate hydrolysis of Ras-bound GTP to GDP, resulting in inactivation of Ras protein. In the present study, we identified two different types of NF1-GRD cDNA. One (type I) is identical to the previously reported sequence, and the other (type II) contained an additional 63 bp insertion that encodes for a region of 21 amino acids in the center of the NF1-GRO molecule. Alternative splicing is the most likely mechanism by which these two types of transcripts arise. Our observations reveal that the type I transcript is predominantly expressed in undifferentiated cells, whereas the type II transcript predominates in differentiated cells. Furthermore, the expression pattern of type I and type II NF1-GRD mRNA immediately changed in SH-SY5Y neuroblastoma cells when neuronal differentiation programs were induced by retinoic acid treatment. We propose that the differential expression of type I and type II NF1-GRD transcripts might be an 'on/off' switch that regulates the catalytic activity of the NF1 gene product, which plays an important role in the regulation of neuronal differentiation.

Immunocytochemical detection of p53 in human gliomas.

Bruner J. M., Saya H., Moser R. P.

Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 4 ( 5 ) 671 - 674 1991.09

ISSN 0893-3952

Since previous published studies of astrocytomas have shown alterations in the short arm of chromosome 17, and this chromosomal location is that which encodes the p53 protein, we used a monoclonal antibody and immunocytochemistry to detect this protein in a series of brain biopsies. The normal p53 protein has a short half-life and is not detectable using this method. Expression of an altered p53 protein was detected in 29 of 71 brain biopsies, but only in those that showed astrocytic features. p53 expression was detected in 20/32 glioblastomas, 5/12 anaplastic astrocytomas, and 3/5 mixed anaplastic oligo-astrocytomas, but only in astrocytic cells. It could not be detected in any other histologic types of primary brain neoplasms, either benign or malignant. The protein was detected in only 1/11 biopsies interpreted as showing gliosis, but this was in a patient who had previously had a resection for glioblastoma, and may have represented unrecognized infiltrating astrocytoma cells. The p53 protein was also expressed in the nuclei of the two human astrocytoma cell lines examined, U251MG and D54MG. These results are in general agreement with previous detailed chromosomal analyses that have found loss of heterozygosity in up to 60% of malignant astrocytic gliomas.

Detection of loss of heterozygosity in the p53 gene in renal cell carcinoma and bladder cancer using the polymerase chain reaction

Oka K., Ishikawa J., Bruner J. M., Takahashi R., Saya H.

Molecular Carcinogenesis 4 ( 1 ) 10 - 13 1991

ISSN 0899-1987

The human p53 gene, a putative tumor suppressor gene, has a polymorphism in amino acid residue 72. We recently developed a method of detecting codon 72 polymorphism in this gene by digestion of polymerase chain reaction-amplified DNA using an allele-specific restriction endonuclease. This polymorphism allows the identification of loss of heterozygosity for the coding region of the p53 gene in limited tissue samples in a short time without using radioactive materials. We examined 33 patients with renal cell carcinoma and 29 with bladder cancer; heterozygosity in the p53 gene was lost in 60% (6 out of 10 cases) and 73% (8 out of 11 cases) of the renal and bladder tumors, respectively. Additionally, the assay's sensitivity could be improved by using DNA extracted from frozen sections of the tumors. Because the proportions of tumor cells and nontumor cells could be assessed by microscopic evaluation of the frozen sections, we were able to minimize contamination from nontumor cells, which occasionally causes false readings of retained heterozygosity. This simple and sensitive method for detecting loss of heterozygosity in the p53 gene makes it possible to rapidly screen a large number of tissue samples and has the potential to be a useful diagnostic tool for a wide variety of human neoplasms.

Pathways of oncogenesis in primary brain tumors

Steck P. A., Saya H.

Current Opinion in Oncology 3 ( 3 ) 476 - 484 1991

ISSN 1040-8746

Codon 72 polymorphism of the TP53 gene

Ara S., Lee P. S Y, Hansen M. F., Saya H.

Nucleic Acids Research 18 ( 16 ) 4961 1990.08

ISSN 0305-1048

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Direct sequencing analysis of transmembrane region of human neu gene by polymerase chain reaction

Saya H., Ara S., Lee P. S Y, Ro J., Hung M. C.

Molecular Carcinogenesis 3 ( 4 ) 198 - 201 1990

ISSN 0899-1987

The neu gene in rat neuro/glioblastoma was found to be activated by a single point mutation in the DNA sequence encoding the transmembrane region of the neu-encoded p185 protein. The human homologue of the rat neu gene, termed c-erbB-2 or HER-2, can also be activated in vitro by a similar mutation in the corresponding region. Although the human neu gene was shown to be amplified/overexpressed in a large portion of human breast and ovarian cancer, no reports indicate that the human neu gene is activated by a point mutation in human tumor. To study the possible point mutation of neu gene in human tumors, we characterized the genomic structure in the transmembrane region of human neu gene, which in turn allowed us to determine DNA sequence in this region directly following DNA amplification by polymerase chain reaction. We analyzed 7 tumor cell lines (2 breast cancer, 1 neuroblastoma, 1 rhabdomyosarcoma, and 3 glioma) and 11 tumor tissue samples (8 breast and 3 ovarian cancers). No mutation was found in the transmembrane region of human neu gene. Our results suggest that unlike the rat neuro/glioblastoma, the single point mutation in the transmembrane region of the human neu gene is a rare event in human tumors. In this study, we developed a technique for direct DNA sequencing of the transmembrane region of the human neu gene. This technique makes it possible to screen a large number of tumor samples.

Modification of a commercially available method of extracting plasmid DNA

Lee P. S Y, Ara S., Saya H.

BioTechniques 8 ( 4 ) 386 1990

ISSN 0736-6205

Application of polymerase chain reaction for rapid subcloning of cDNA inserts from λgt11 clones

Herrmann J., Lee P., Saya H., Nakajima M.

BioTechniques 8 ( 4 ) 376 - 381 1990

ISSN 0736-6205

Cell kinetic analysis of human brain tumors by bivariate flowcytometric measurement of cellular DNA content and amount of incorporated bromodeoxyuridine

Okuda Y., Taomoto K., Saya H., Ijichi A., Kudo H., Kokunai T., Tamaki N., Matsumoto S.

Brain and Nerve 41 ( 4 ) 383 - 390 1989

ISSN 0006-8969

Antigen common to several species, recognized by a rat monoclonal antibody raised against syngeneic rat bladder tumor

Eto H., Saya H., Nakata M., Mizoguchi A., Kamidono S.

International Journal of Cancer 44 ( 3 ) 454 - 459 1989

ISSN 0020-7136

A rat monoclonal antibody (MAb) termed RS-II (Ig M) was obtained by syngeneic immunization with rat bladder tumor cells induced by N-butyl, N-hydroxybutylnitrosamine (BBN). Immunocytochemical analysis showed that RS-II is also reactive with mouse, dog and human bladder tumor-cell lines and some other human tumor-cell lines but not myeloma or leukemia cells. Immunohistochemical examination of paraffin-embedded tissues has shown that RS-II is reactive with mouse, rat, dog and human bladder tumors (5/5, 5/5, 1/1, 31/49) and some other tumors, but not with normal human urothelium or normal rat tissues. The antigen is expressed on the majority of low-grade or well-differentiated tumors, but less on advanced, invasive tumors. The immunofluorescence staining pattern of cultured cells was cytoplasmic and filamentous. Immunoblotting revealed that the antigen is a M(r) 54,000 and M(r) 56,000 protein which was extracted with difficulty from the cultured cells with Triton X-100. The antigen was also detected in the culture supernatant by means of ELISA. Our results suggest that the epitope is expressed on cytoskeletons common to a wide variety of mammalian cells at a certain stage of differentiation.

Neuronal cell differentiation of human neuroblastoma cells by retinoic acid plus herbimycin A

Preis P. N., Saya H., Nadasdi L., Hochlaus G., Levin V., Sadee W.

Cancer Research 48 ( 22 ) 6530 - 6534 1988

ISSN 0008-5472

We investigated the effect of retinoic acid (RA) and herbimycin A (herb-A) on cell growth, cell differentiation, and colony formation of human neuroblastoma cell lines. The neuroblastoma line SK-N-SH expressed both neuroblast and nonneuronal phenotypes, whereas its subclone SH-SY5Y and the Kelly cell line were predominantly neuroblastic. Both herb-A and RA, given alone, moderately reduced cell growth and colony formation of the neuroblastic cell lines. Growth curve analyses with SK-N-SH suggested that herb-A greatly reduced the number of initially growing cells, whereas RA slightly enhanced initial cell growth. Morphological changes were determined with the use of rhodamine-phalloidin staining of actin. Retinoic acid caused an increase in the fraction of neuroblast cell in SK-N-SH, and conversely of nonneuronal cells in SH-SY5Y and Kelly cell lines. Both drugs also caused partial differentiation towards a neuronal phenotype, and herb-A induced selective lysis of nonneuronal cells of SK-N-SH. Because of their discrepant effects, RA (10 μM) and herb-A (236 nM) were tested in combination at a concentration that had only moderate effects when given alone. The combination further reduced cell growth and colony formation and dramatically enhanced differentiation towards a neuronal morphology. The Kelly cell line with amplified N-myc genome, which correlates with clinical progression of neuroblastoma, was also sensitive to RA plus herb-A. These results recommend the combination of RA and herb-A for differentiation therapy of neuroblastoma.

A hypothesis for myeloschisis

Oi S., Saya H., Matsumoto S.

Journal of Neurosurgery 68 ( 6 ) 947 - 954 1988

ISSN 0022-3085

A hypothesis for embryopathogenesis of myeloschisis is described on the basis of experimental studies analyzing the stage specificity and immunohistochemical/histological characteristics of the exposed neural tissue (placode). Myeloschisis developed in six fetuses among 205 chick embryos treated in various stages with teratogens including ethylnitrosourea, and anticonvulsant and antipyretic agents. All but one case (with associated cephalothoracopagus) demonstrated myeloschisis in the thoracic region with a lamina defect of two and three levels. No fetus was exposed to a teratogen prior to or within Hamburger and Hamilton stage 12 (45 to 49 hours postincubation), when the neuropore closes. Immunohistochemical studies of chick myeloschisis clearly indicated that neuron-specific enolase-positive elements were extremely active only in the overgrown placode, corresponding to the histological findings with Kluver-Barrerra's special stain. These findings were compared with observations in a case of myeloschisis in a human neonate. The results of this study imply the possibility of another mechanism for the embryopathogenesis of myeloschisis: namely, the overgrowth and reopening hypothesis.

Pineoblastoma with neuronal differentiation. Immunohistochemical and immunocytochemical studies

Okuda Y., Taomoto K., Saya H., Ijichi A., Kokunai T., Tamaki N., Matsumoto S.

Journal of Neuro-Oncology 6 ( 2 ) 193 - 198 1988

ISSN 0167-594X

Protein-tyrosine kinase activity and pp60(v-src) expression in whole cells measured by flow cytometry

Preis P. N., Waldman F. M., Frackelton A. R., Saya H., Levin V. A.

Cancer Research 48 ( 16 ) 4633 - 4638 1988

ISSN 0008-5472

The expression and phosphotyrosine activity of pp60(v-src) were measured in the B31 avian sarcoma virus-transformed rat cell line by flow cytometry using monoclonal antibodies against pp60(v-src) (EB7) and phosphotyrosine (1G2). Although the immunocytochemical staining was markedly heterogeneous, binding of both antibodies was significantly greater to B31 cells than to untransformed Rat 1 cells. Binding of 1G2 to phosphotyrosine residues was specific; it was entirely inhibited by adding excess phenylphosphate but was not affected by phosphoserine or phosphothreonine. The relationship between the amount of phosphorylated tyrosine measured by phosphoamino acid analysis was linear in vanadate-treated BALB/c 3T3 cells. Treatment of B31 cells for 48 h with herbimycin A, a benzenoid ansamycin antibiotic, to decrease the expression and tyrosine kinase activity of pp60(v-src) caused reductions of 42% in anti-pp60(v-src) and 58% in anti-phosphotyrosine antibody immunofluorescence. DNA staining with the fluorescent dye propidium iodide showed no cell cycle specificity in the binding of either antibody. Herbimycin A also caused the transformed cell line to revert to the morphology, actin configuration, and growth behavior of untransformed cells; these changes were reversed within 12 h after removal of the drug. Flow cytometric evaluation of tyrosine kinase expression and activity was fast and easy, and the results correlated well with other measures of cell phenotype. This technique can be used to quantitate the effects of drugs on oncogenic proteins such as pp60(v-src) and their associated tyrosine kinase activity.

Detection of human glioma-associated antigen by rat monoclonal anibody raised against syngeneic rat glioma cells

Saya H., Masuko T., Kokunai T., Yagita H., Ijichi A., Taomoto K., Tamaki N., Matsumoto S., Hashimoto Y.

Journal of Neurosurgery 65 ( 4 ) 495 - 502 1986

ISSN 0022-3085

A monoclonal antibody termed 'FR77' was obtained from a hybridoma clone established by fusion between P3x63Ag8.653 mouse myeloma cells and spleen cells of a Fischer F344 rat hyperimmune to syngeneic 9L/R₃ glioma cells. Immunoperoxidase staining of various cultured cells showed that FR77 was reactive to both rat and human glioma cells, but was not reactive with other nonglioma cells. Immunohistochemical examination of paraffin-embedded or cryostat-frozen sections of various human tissues revealed that FR77 was strongly reactive with glioblastoma, grade III astrocytoma, and craniopharyngioma; partially reactive with intracerebral primitive neuroectodermal tumor, pineoblastoma, and desmoplastic medulloblastoma; and weakly reactive with low-grade astrocytoma. It was not reactive with other types of brain tumors and normal human tissues tested. The FR77-defined antigen was observed to be predominantly localized in the cytoplasm of antigen-bearing cells as suggested by the immunostaining pattern, but part of it was also expressed on the cell surface of glioma cells as demonstrated by a complement-mediated cytotoxic test. Fractionation of the antigenic component and periodic acid treatment of tumor tissue bearing the FR77-defined antigen indicated that the antigen is of a neutral glycolipid nature and that the antigenic determinant to FR77 is present on its sugar portion.

Derivation of a brain tumor-selective monoclonal antibody from hybridoma between mouse myeloma and rat spleen cells immune to syngeneic glioma

Saya Hideyuki, Masuko Takashi, Hashimoto Yoshiyuki

Japanese Journal of Cancer Research 76 ( 12 ) 1198 - 1202 1985.12

ISSN 0910-5050

Fischer 344 (F344) rats hyperimmunized with syngeneic 9L R₈ glioma cells produced antibody selective to glioma cells. Hybridomas prepared from the spleen cells of the immunized rat were cloned, and we obtained a hybridoma clone which produced monoclonal IgM antibody, termed FR77, that showed selectivity to glioma cells. Immunoperoxidase staining of cultured cells revealed that FR77 was reactive with 3 lines of rat glioma cells but not with normal F344 rat fibroblasts. Immuno-histochemical staining of F344 rat tissue sections with biotinylated FR77 demonstrated that FR77 could bind with glioma tissue developed by intracerebral injection of 9L R₈ glioma cells but not to normal parts of the brain tissues and other normal tissues tested. The FR77-defined antigen was observed to be mainly localized in cytoplasm of glioma cells but a small portion of the antigen was also detected on the glioma cell surface.

Angiographic classification of aneurysms of the horizontal segment of the anterior cerebral artery

Wakabayashi Toshimitsu, Tamaki Norihiko, Yamashita Hideyuki, Saya Hideyuki, Suyama Tohru, Matsumoto Satoshi

Surgical Neurology 24 ( 1 ) 31 - 34 1985

ISSN 0090-3019

Ten aneurysms of the horizontal portion of the anterior cerebral artery (A₁) were analyzed. These 10 aneurysms were classified into three types according to the mode of the origin of the neck of the aneurysm from the A₁ segment: (a) an aneurysm originating from the junction of the A₁ segment and the lenticulostriate artery, (b) one from the proximal end of the fenestration of the A₁ and (c) one from the top of the A₁ loop (elongation and kinking of the A₁ segment). In these aneurysms, their size, the side of the A₁ segment with an aneurysm, the multiplicity of aneurysms, the coexistence of hypertension, the grade of the patients, the laterality of the subarachnoid blood clots (as seen on computed tomography scans) and surgical outcome were analyzed.

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